1All Fermentas products are manufactured in class D clean room facilities, qualified and certified as per EU directives and ISPE guidelines. Fermentas quality assurance is carried out according to ISO9001 quality and ISO14001 environmental management systems, guaranteeing batch-to-batch reproducibility. Integration of our clean room and ISO systems ensures stability and the absence of contaminants in all of our products.
FastDigest® and Conventional Restriction Enzymes .................................................. 2PureExtreme® Quality ...................................................................................................... 2Labeled Oligonucleotide (LO) Test .................................................................................. 2Navigation Guide .............................................................................................................. 3Description of Icons ......................................................................................................... 4
FastDigest® Restriction Enzymes .................................................................................... 5Activity and Quality Control Assays ............................................................................... 6Storage and Shipping ...................................................................................................... 6Product List ...................................................................................................................... 7Product Description ....................................................................................................... 11Protocols and Recommendations ................................................................................. 70
1.1. Fast DNA digestion ................................................................................................ 701.2. Reaction set-up for digestion of multiple DNA samples ........................................... 701.3. Double and multiple digestion of DNA ................................................................... 701.4. Scaling up DNA digestion reaction ......................................................................... 70
Reaction Conditions ....................................................................................................... 71Conventional Restriction Enzymes ............................................................................... 75
Activity and Quality Control Assays ............................................................................. 75Storage and Shipping .................................................................................................... 75Product List and Cross-reference to FastDigest® Restriction Enzymes ................... 76Product Description ....................................................................................................... 80
Nicking Enzymes ....................................................................................................... 157Homing Enzyme ......................................................................................................... 159
Protocols and Recommendations ............................................................................... 1601.5. DNA digestion .................................................................................................... 1601.6. Digestion of PCR products .................................................................................. 1601.7. Setting up double digestion ................................................................................. 1601.8. Stability during prolonged incubation ................................................................... 1601.9. Dilution of restriction enzymes ............................................................................. 1601.10. Partial digestion of DNA..................................................................................... 1601.11. Digestion of agarose-embedded DNA ................................................................. 1611.12. Inactivation of restriction enzymes ..................................................................... 161
Reaction Conditions .................................................................................................... 162Reaction Buffers ........................................................................................................ 162Recommended Reaction Conditions ........................................................................... 163Activity of Mesophilic and Thermophilic Enzymes at 37°C ........................................... 167Double Digestion in Universal Tango™ Buffer............................................................... 168Digestion of Agarose-Embedded DNA ........................................................................ 170Cleavage Efficiency Close to the Termini of PCR Fragments ......................................... 171Cleavage of Restriction Targets Located in Close Vicinity within pUC19 Multiple Cloning Site ............................................................................................................... 172
Common Properties ........................................................................................................ 173Classification of Restriction Enzymes ....................................................................... 173Site Preferences by Restriction Endonucleases........................................................ 173Star Activity ...................................................................................................................174Digestion of Methylated DNA ...................................................................................... 175
Effect of Dam Methylation on DNA Cleavage ............................................................... 176Effect of Dcm Methylation on DNA Cleavage ............................................................... 177Effect of CpG Methylation on DNA Cleavage ............................................................... 178Effect of EcoKI and EcoBI Methylation on DNA Cleavage ............................................. 181
Newly Generated Cleavage Sites ................................................................................ 182Recognition Sites Resulting from Ligation of Blunt DNA Ends ....................................... 182Recognition Sites Resulting from Ligation of Protruding Compatible DNA Ends ............. 192Recognition Sites Resulting from Fill-in of 5’-overhang and Self-ligation....................... 202Recognition Sites Resulting from Removal of 3’-overhang and Self-ligation ..................206
Selection Guides .............................................................................................................. 207Alphabetic List of Commercially Available Restriction Enzymes ............................ 207Alphabetic List of Recognition Sequences ................................................................ 219Enzyme Recognizing 2 bp Long Targets ....................................................................222Alphabetic List of Enzymes Recognizing 4 bp Long Targets ....................................222Alphabetic List of Enzymes Recognizing 5 bp Long Targets ....................................222Alphabetic List of Enzymes Recognizing 6 bp Long Targets .................................... 223Alphabetic List of Enzymes Recognizing 7 bp Long Targets .................................... 224Alphabetic List of Enzymes Recognizing 8 bp Long Targets .................................... 224Commercial Restriction Enzymes Generating 5’-protruding Ends .......................... 225Commercial Restriction Enzymes Generating 3’-protruding Ends .......................... 228Commercial Restriction Enzymes Generating Blunt Ends ........................................ 230Commercial Restriction Enzymes Cleaving DNA on the Both Sides of their Recognition Sequence ................................................................................................. 230
Troubleshooting Guide ................................................................................................... 231
Fast
Dige
st® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
2
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Restriction enzymes recognize specific nucleo-tide sequences and cleave DNA molecules at a position either within or outside their recognition site. These enzymes are important tools for numerous applications, including studies of DNA primary structure and recombinant DNA tech-nology. The best studied and the most widely
used are type II restriction endonucleases. More than 3800 type II restriction enzymes, exhibiting almost 290 different specificities, have been isolated. Since 1977 Fermentas has discovered approxi-mately 30% of all known restriction enzymes. We are a leading global enzyme manufacturer,
Labeled Oligonucleotide (LO) TestThe Labeled Oligonucleotide Test was designed at Fermentas for detection of trace contaminat-ing activities in enzyme, nucleotide and reagent solutions. This unique test allows us to detect trace activities of endo-, exodeoxyribonucleases and phosphatases in enzyme preparations. Therefore we are able to bring to the market only highest quality and performance products.Single-stranded and double-stranded 5’-[32P]-labeled synthetic oligonucleotides containing no recognition sites for restriction enzymes are used as substrates for the LO test. The labeled oligonucleotides are incubated with excess of restriction enzyme, separated on a polyacrylamide gel under denaturing conditions and analyzed by phospho-imaging. The presence of contaminating endo- and exodeoxyribonucleases results in the degrada-tion of the labeled substrates (see Fig. 1.1), while decreased specific radioactivity indicates the presence of contaminant phosphatases. The restriction enzyme passes this quality control test if there is neither degradation of labeled oligonucleotides nor a decrease in specific radioactivity.
supplying both conventional restriction enzymes and innovative restriction enzyme line for rapid DNA digestion in one universal buffer – FastDigest® restriction enzymes. Fermentas offers 176 FastDigest® and 201 conventional restriction enzymes.
Figure 1.1. Labeled Oligonucleotide (LO) Test.ss – single-stranded radiolabeled oligonucleotideds – double-stranded radiolabeled oligonucleotidePure enzyme – Fermentas NotIContaminated enzyme – competitor’s NotI
Labeled Oligonucleotide Test
Radiolabeled ss and ds oligonucleotides mixed with an excess of enzyme
Incubated at 37°C for 4 hours
Denatured and separated by PAGE
Band pattern imaged and analyzed
Patterns Typical for:
Control Pure enzyme
Contaminated enzyme
ss ds ss ds ss ds
Degradation due to contamination with non-specific nucleases
PureExtreme® Quality
Fermentas Restriction enzymes are produced in clean room class D facilities under the ISO 9001:2008 quality management system and are subjected to extensive quality control.
Fermentas restriction enzymes pass all industry standard quality control assays, as well as the unique Labeled Oligonucleotide
(LO) Test, which is the most sensitive test for the detection of trace activities of endodeoxy-ribonucleases, exodeoxyribonucleases and phosphatases see Fig. 1.1.A warranty is assigned and an expiry date is listed both on the product label and in the Cer-tificate of Analysis supplied with each product. Product lots are monitored to meet the quality specifications up to the expiry date.
FastDigest® and Conventional Restriction Enzymes
3
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Navigation GuideTable 1.1. FastDigest® and conventional restriction enzymes.To find or select a restriction enzyme Page
By name
Table 1.2. FastDigest® restriction enzymes 7
Table 1.4. Fermentas conventional restriction enzymes 76
Table 1.25. Commercially available restriction enzymes 207
On-line. REsearch™ at www.fermentas.com/research
By recognition sequence
Table 1.26. Recognition sequences of restriction enzymes 219
Table 1.27. Enzyme recognizing 2 bp long targets 222
Table 1.28. Enzymes recognizing 4 bp long targets 222
Table 1.29. Enzymes recognizing 5 bp long targets 222
Table 1.30. Enzymes recognizing 6 bp long targets 223
Table 1.31. Enzymes recognizing 7 bp long targets 224
Table 1.32. Enzymes recognizing 8 bp long targets 224
On-line. REsearch™ at www.fermentas.com/research
By number of recognition sites in DNA
Table 14.15. Number of recognition sites in DNA molecules 514
Appendix. Phage and plasmid DNA. 489
On-line. REviewer™ at www.fermentas.com/reviewer
By type of generated DNA ends
Table 1.33. Commercial restriction enzymes generating 5’-protruding ends 225
Table 1.34. Commercial restriction enzymes generating 3’-protruding ends 228
Table 1.35. Commercial restriction enzymes generating blunt ends 230
Table 1.36. Commercial restriction enzymes cleaving DNA on the both sides of their recognition sequence 230
On-line. REsearch™ at www.fermentas.com/research
By cleavage efficiency close to termini of DNA
Table 1.3. Reaction conditions for FastDigest® restriction enzymes 71
Table 1.10. Cleavage efficiency close to the termini of PCR fragments 171
Table 1.11. Cleavage of restriction targets located in close vicinity within pUC19 multiple cloning site 172
By sensitivity to DNA methylation
Table 1.12. Fermentas isoschizomers and neoschizomers with differing sensitivities to target methylation 175
Tables 1.13, 1.14. Effect of Dam methylation 176
Tables 1.15, 1.16. Effect of Dcm methylation 177
Tables 1.17, 1.18. Effect of CpG methylation 178
Tables 1.19, 1.20. Effect of EcoBI and EcoKI methylation 181
By newly generated recognition sites
Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends 182
Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends 192
Table 1.23. Newly generated recognition sites resulting from fill-in of 5’-overhang and self-ligation 202
Table 1.24. Newly generated recognition sites resulting from removal of 3’-overhang and self-ligation 206
To perform fast digestion of DNA in universal FastDigest® buffer
Fast DNA digestion reaction set-up Protocols and recommendations for fast DNA digestion 70
Digestion of different DNA substrates
Table 1.3. Reaction conditions for FastDigest® restriction enzymes 71Digestion close to DNA terminiInactivation of enzyme after digestionIncubation time without star activityCompatibility of FastDigest® enzyme buffer with downstream applications 2.1. Activity of DNA/RNA modifying enzymes in Fermentas buffers 274
Troubleshooting Table 1.37. Troubleshooting guide for DNA digestion 232
To perform DNA digestion with conventional restriction enzymesDNA digestion reaction set-up Protocols and recommendations for DNA digestion with conventional restriction enzymes 160
Double/multiple digestion of DNAOn-line. DoubleDigest™ at www.fermentas.com/doubledigest
Table 1.8. Double digestion in universal Tango™ buffer 168
Table 1.6. Reaction conditions for restriction enzymes 163
Reaction conditions for individual enzymesTable 1.6. Reaction conditions for restriction enzymes 163
Table 1.5. Reaction buffers for restriction enzymes 162
Digestion close to DNA terminiTable 1.10. Cleavage efficiency close to the termini of PCR fragments 171
Table 1.11. Cleavage of restriction targets located in close vicinity within pUC19 multiple cloning site 172
Digestion of agarose-embeded DNA 1.11 Table 1.9. Digestion of agarose-embedded DNA 161
Inactivation of enzyme after digestion1.12. Inactivation of restriction enzymes 161
Table 1.6. Reaction conditions for restriction enzymes 163
How to identify and avoid star activity Description of star activity of restriction enzymes 174
Compatibility of conventional restriction enzyme buffers with downstream applications 2.1. Activity of DNA/RNA modifying enzymes in Fermentas buffers 274
Partial digestion of DNA 1.10. Partial digestion of DNA 160
How to dilute restriction enzymes 1.9. Dilution of restriction enzymes 160
Stability during prolonged incubation Table 1.6. Reaction conditions for restriction enzymes 163
Enzyme activity at non-optimal temperature Table 1.7. Activity of mesophilic and thermophilic enzymes at 37°C 167
Troubleshooting guide Table 1.37. Troubleshooting guide for DNA digestion 232
4
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Description of Icons
Additives. Indicates additives required to obtain the stated enzyme activity. Solutions of S-adenosyl-methionine and oligonucleotides are supplied with enzymes. DTT (#R0861) is available separately (p.476).
Incubation Temperature. Indicates the optimal incubation temperature in degree Celsius (°C).
Ligation Efficiency. Indicates the ligation efficiency of DNA fragments generated by digestion with the restriction enzyme (see p.6 and p.75).
Star Activity. Indicates restriction enzymes prone to star activity (see p.174).
Sensitivity to Methylation. DNA cleavage by the restriction enzyme is blocked or impaired by Dam, Dcm or CpG methylation within the target sequences (see p.175).
High Concentration. Indicates enzymes available at a high concentration (50 u/µl).
Genome Qualified. Indicates that the restriction enzyme cleaves agarose-embedded DNA (see p.161).
Recombinant Enzyme. Indicates enzymes purified from recombinant E.coli.
Blue/White Certified. Enzyme was tested by the Blue/White cloning assay (see p.6 and p.75).
FastDigest® Enzyme. Enzyme is available in FastDigest® format (see p.5).
Buffers. Letters in the buffer icon indicate the recommended buffer for a specific restriction enzyme. FastDigest® and FastDigest® Green Buffers are indicated by “F” icon. B (blue), G (green), O (orange), R (red) and Tango™ (yellow) correspond to the color codes of the Five Buffer System for conventional restriction enzymes. The “Unique” icon indicates conventional restriction enzymes that require a special buffer, which is supplied with the enzyme (see p.162).
Thermal Inactivation. Indicates conditions for thermal inactivation of the enzyme (see p.161).Indicates that only small amounts of restriction enzyme (up to 10 units) can be thermally inactivated.
LO Certified. Enzyme was tested by the labeled oligonucleotide (LO) assay (see p.2 and p.75).
Sensitivity to Overlapping Dam, Dcm or CpG Methylation. The target site may be methylated in certain sequence contexts (overlapping methylation). This will result in blocked or impaired DNA cleavage (see p.175).
Single letter codeR = G or A; H = A, C or T; Y = C or T; V = A, C or G;W = A or T; B = C, G or T;M = A or C; D = A, G or T;K = G or T; N = G, A, T or C.S = C or G;
5
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
DescriptionFastDigest® enzymes are an advanced line of restriction enzymes for rapid DNA digestion. All FastDigest® enzymes are 100% active in the universal FastDigest® and FastDigest® Green buffers and are able to digest DNA in 5-15 min-utes. This enables any combination of restriction enzymes to work simultaneously in one reaction tube and eliminates the need for sequential digestions. As an added convenience, the Fast-Digest® Green Buffer allows for direct loading of the reaction mixture on a gel.FastDigest® enzymes can be used to digest plasmid, genomic and viral DNA as well as PCR products and do not show star activity even in prolonged incubations.The FastDigest® line is ideal for use in applications that require high purity reaction components, performance reliability and simple reaction set-up.
Universal FastDigest® BufferTwo versions of the universal FastDigest® buffer are supplied with each enzyme: 10X FastDi-gest® Buffer and 10X FastDigest® Green Buffer. All FastDigest® enzymes are 100% active in both FastDigest® and FastDigest® Green buffers. Enzymes used in common downstream applications such as ligation, blunting and dephosphorylation also have 100% activity in both buffers. As an added convenience, the FastDigest® Green Buffer contains a density reagent and two tracking dyes that allow for direct loading of the reaction mixture on gels. The blue dye migrates with 3-5 kb DNA fragments in 1% agarose gel and has an excitation peak at 424 nm while the yellow dye migrates faster than 10 bp DNA fragments in 1% agarose gel and has an excitation peak at 615 nm. The presence of the dyes in the FastDigest® Green buffer does not interfere with DNA digestion or with downstream applications but may hinder digestion product analysis by fluorescence excitation. For such applications we recommend use of the colorless FastDigest® Buffer.
Features• 100% activity of all FastDigest® enzymes in
the new green universal digestion buffer.• 100% buffer compatibility with all down-
stream applications.• Complete digestion in 5-15 minutes.• Direct loading of reaction mixture on gels.
Applications• Fast clone analysis.• Fast preparation of DNA for cloning.• Digestion of PCR products.• Fast RFLP genotyping.• Digestion of difficult-to-cleave DNA.
FastDigest® Restriction Enzymes
M C 1 2 C 3 4 M
FastDigest® Buffer
FastDigest® GreenBuffer
Figure 1.2. Five minute triple digestion followed by ligation of plasmid DNA in the universal
FastDigest® and FastDigest® Green buffers.
M – GeneRuler™ Express DNA Ladder (#SM1551).C – undigested plasmid DNA control.1 – plasmid DNA triple digested with FastDigest® EcoRI, FastDigest® KpnI and FastDigest® SmaI in FastDigest® Buffer.2 – the reaction mixture after ligation in FastDigest® Buffer.3 – plasmid triple digested with FastDigest® EcoRI, FastDigest® KpnI and FastDigest® SmaI in FastDigest® Green Buffer.4 – the reaction mixture after ligation in FastDigest® Green Buffer.
1 2
Figure 1.3. FastDigest® Green Buffer.1 – Reaction mixture containing FastDigest® Green Buffer before electrophoresis.2 – Reaction mixture containing FastDigest® Green Buffer after electrophoresis.
6
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Activity and Quality Control Assays
Activity Assay1 µl of FastDigest® enzyme is formulated to cleave 1 µg of substrate DNA in 5 or 15 min at recommended temperature in 1X FastDigest® buffer. 1 µl of FastDigest® restriction endonu-clease corresponds to 1 FastDigest® unit (FDU) of enzyme. In general, enzymes are assayed with λ phage DNA at 37°C. However, some exceptions apply:
• Restriction enzymes that show optimum activity at temperatures other than 37°C are assayed under their optimal temperature.
• FastDigest® enzymes that do not have recog-nition sites on λ DNA are assayed with other specific DNA substrates, as indicated in the Certificates of Analysis.
• FastDigest® enzymes with only a few recog-nition sites on the λ DNA are assayed using λ DNA cut with another restriction enzyme.
• FastDigest® enzymes sensitive to Dam or Dcm methylation are assayed using DNA purified from dam–, dcm– strain of E.coli.
Quality Control
Labeled Oligonucleotide (LO) Test
1µl of FastDigest® enzyme is incubated with 5’-[32P]-labeled single-stranded and double-stranded synthetic oligonucleotides in 1X FastDigest® buffer for 1 hour at the recommended temperature. The restriction enzyme passes this quality control test if there is no degradation of labeled oligonucleotides and no decrease in their specific radioactivity.
Prolonged Incubation / Star Activity AssayThe star activity assay is designed to test alterations in the DNA digestion pattern after prolonged incubation with FastDigest® enzymes.1 µl of FastDigest® enzyme is incubated with 1 µg of substrate DNA at the recommended temperature for up to 16 hours. After electro-phoretic separation of the DNA fragments, the characteristic banding patterns are examined for alterations. The maximal incubation time that does not cause any star activity is indicated for each FastDigest® enzyme in the product description and the Certificate of Analysis sup-plied with each enzyme.
Ligation and Recleavage AssayThe ligation and recleavage assay confirms the integrity of DNA ends. DNA fragments obtained after overdigestion with 1 µl of FastDigest® enzyme for 1 hour are ligated with T4 DNA ligase and then recut with the same enzyme. A FastDigest® restriction enzyme conforms to the quality criterion if the observed ligation efficiency is identical to that of conventional enzyme.The percentage of DNA that can be successfully ligated and then re-cleaved for each FastDigest® restriction enzyme is indicated in the in the pro-duct description and the Certificate of Analysis supplied with each enzyme.
Blue/White (B/W) Cloning AssayThe Blue/White cloning assay is designed to test the integrity of DNA ends. pUC57 DNA is digested at unique sites within the lacZ reporter gene with 1 µl of a FastDigest® restriction enzyme in 1X FastDigest® buffer. After a 1 hour incubation, the plasmid DNA is recircularized by ligation and transformed into E.coli XL1-Blue competent cells. The cells are then plated onto X-Gal/IPTG/Amp agar. An intact lacZ gene will give rise to a blue colony. If the termini of the linearized pUC57 are altered by contaminating exodeoxynucleases, the lacZ reading frame is interrupted, which results in the appearance of white colonies. An enzyme conforms to this quality criterion if the number of white colonies does not exceed 3%. For FastDigest® restric-tion enzymes lacking recognition sites in pUC57 DNA, the assay is performed with the mixture of pUC57/HindIII, pUC57/PstI and pUC57/Eco32I DNA fragments representing three different types of termini (3’-overhang, 5’-overhang and blunt ends).
Storage and ShippingAll FastDigest® restriction enzymes should be stored at -20°C. During shipment on dry ice, enzymes may freeze. This does not affect their quality – all Fermentas enzymes are 100% active after at least three freeze-thaw cycles.For 24-48 hour delivery, enzymes may be shipped on blue ice since their quality is not affected by short exposure to 4°C.
7
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
(continued on next page)
Product List
Table 1.2. FastDigest® restriction enzymes.
FastDigest® restriction enzyme
Prototype Specificity 5’→3’ Cat. # Page
FastDigest® AatII AatII GACGT↓C FD0994 11FastDigest® AccI (XmiI) AccI GT↓MKAC FD1484 11FastDigest® Acc65I KpnI (GGTAC↓C) G↓GTACC FD0904 11FastDigest® AciI (SsiI) AciI CCGC(-3/-1)↓ FD1794 12FastDigest® AclI (Psp1406I) AclI AA↓CGTT FD0944 12FastDigest® AcuI (Eco57I) Eco57I CTGAAG(16/14)↓ FD0344 12FastDigest® AfeI (Eco47III) Eco47III AGC↓GCT FD0324 13FastDigest® AflII (BspTI) AflII C↓TTAAG FD0834 13FastDigest® AgeI (BshTI) AgeI A↓CCGGT FD1464 13FastDigest® AjuI AjuI ↓(7/12)GAA(N)7TTGG(11/6)↓ FD1954 14FastDigest® AleI (OliI) OliI CACNN↓NNGTG FD1634 14FastDigest® AluI AluI AG↓CT FD0014 14FastDigest® Alw21I HgiAI GWGCW↓C FD0024 15FastDigest® Alw26I BsmAI GTCTC(1/5)↓ FD0034 15FastDigest® AlwNI (CaiI) AlwNI CAGNNN↓CTG FD1394 15FastDigest® ApaI ApaI GGGCC↓C FD1414 16FastDigest® ApaLI (Alw44I) ApaLI G↓TGCAC FD0044 16FastDigest® AscI (SgsI) AscI GG↓CGCGCC FD1894 16FastDigest® AseI (VspI) VspI AT↓TAAT FD0914 17FastDigest® AsiSI (SfaAI) SgfI GCGAT↓CGC FD2094 17FastDigest® AvaI (Eco88I) AvaI C↓YCGRG FD0384 17FastDigest® AvaII (Eco47I) AvaII G↓GWCC FD0314 18FastDigest® AvrII (XmaJI) AvrII C↓CTAGG FD1564 18FastDigest® BamHI BamHI G↓GATCC FD0054/5 18FastDigest® BanI (BshNI) HgiCI G↓GYRCC FD1004 19FastDigest® BbsI (BpiI) BbvII GAAGAC(2/6)↓ FD1014 19FastDigest® BbvI (Lsp1109I) BbvI GCAGC(8/12)↓ FD2074 19FastDigest® BclI BclI T↓GATCA FD0724 20FastDigest® BfaI (FspBI) MaeI C↓TAG FD1764 20FastDigest® BglI BglI GCCNNNN↓NGGC FD0074 20FastDigest® BglII BglII A↓GATCT FD0083/4 21FastDigest® BlpI (Bpu1102I) EspI GC↓TNAGC FD0094 21FastDigest® Bme1580I (BseSI) BseSI GKGCM↓C FD1444 21FastDigest® BmtI (BspOI) NheI (G↓CTAGC) GCTAG↓C FD2044 22FastDigest® BplI BplI ↓(8/13)GAG(N)5CTC(13/8)↓ FD1314 22FastDigest® BpmI (GsuI) BpmI CTGGAG(16/14)↓ FD0464 22FastDigest® Bpu10I Bpu10I CCTNAGC(-5/-2)↓ FD1184 23FastDigest® BsaAI (Ppu21I) BsaAI YAC↓GTR FD1974 23FastDigest® BsaBI (BseJI) BsaBI GATNN↓NNATC FD1414 23FastDigest® BsaHI (Hin1I) AcyI GR↓CGYC FD0474 24FastDigest® BsaJI (BseDI) SecI C↓CNNGG FD1084 24FastDigest® BseGI FokI (GGATG(9/13)↓) GGATG(2/0)↓ FD0874 24FastDigest® BseNI BsrI ACTGG(1/-1)↓ FD0884 25FastDigest® BseXI BbvI GCAGC(8/12)↓ FD1454 25FastDigest® Bsh1236I FnuDII CG↓CG FD0924 25FastDigest® BsiEI (Bsh1285I) McrI CGRY↓CG FD0894 26FastDigest® BsiWI (Pfl23II) SplI C↓GTACG FD0854 26FastDigest® BslI (BseLI) BsiYI CCNNNNN↓NNGG FD1204 26
8
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.2. FastDigest® restriction enzymes.
(continued on next page)
FastDigest® restriction enzyme
Prototype Specificity 5’→3’ Cat. # Page
FastDigest® BsmBI (Esp3I) Esp3I CGTCTC(1/5)↓ FD0454 27FastDigest® BsmFI (FaqI) FinI GGGAC(10/14)↓ FD1814 27FastDigest® Bsp119I AsuII TT↓CGAA FD0124 27FastDigest® Bsp120I ApaI (GGGCC↓C) G↓GGCCC FD0134 28FastDigest® Bsp1286I (SduI) SduI GDGCH↓C FD0654 28FastDigest® Bsp1407I Bsp1407I T↓GTACA FD0933/4 28FastDigest® BspCNI (BseMII) BseMII CTCAG(10/8)↓ FD1404 29FastDigest® BspHI (PagI) BspHI T↓CATGA FD1284 29FastDigest® BspMI (BveI) BspMI ACCTGC(4/8)↓ FD1744 29FastDigest® BsrBI (MbiI) BsrBI CCGCTC(-3/-3)↓ FD1274 30FastDigest® BsrDI (BseMI) BsrDI GCAATG(2/0)↓ FD1264 30FastDigest® BsrFI (Cfr10I) Cfr10I R↓CCGGY FD0184 30FastDigest® BssHII (PteI) BsePI G↓CGCGC FD2134 31FastDigest® BstXI BstXI CCANNNNN↓NTGG FD1024 31FastDigest® BstZ17I (Bst1107I) SnaI GTA↓TAC FD0704 31FastDigest® Bsu36I (Eco81I) SauI CC↓TNAGG FD0374 32FastDigest® ClaI (Bsu15I) ClaI AT↓CGAT FD0143/4 32FastDigest® Csp6I RsaI (GT↓AC) G↓TAC FD0214 32FastDigest® DdeI (HpyF3I) DdeI C↓TNAG FD1884 32FastDigest® DpnI DpnI Gm6A↓TC FD1703/4 33FastDigest® DraI AhaIII TTT↓AAA FD0224 33FastDigest® DraIII (AdeI) DraIII CACNNN↓GTG FD1234 34FastDigest® DrdI (AasI) DrdI GACNNNN↓NNGTC FD1724 34FastDigest® EagI (Eco52I) XmaIII C↓GGCCG FD0334 34FastDigest® Eam1105I Eam1105I GACNNN↓NNGTC FD0244 35FastDigest® EarI (Eam1104I) Ksp632I CTCTTC(1/4)↓ FD0234 35FastDigest® Ecl136II SacI (GAGCT↓C) GAG↓CTC FD0254 35FastDigest® Eco31I Eco31I GGTCTC(1/5)↓ FD0293/4 36FastDigest® Eco91I BstEII G↓GTNACC FD0394 36FastDigest® EcoNI (XagI) EcoNI CCTNN↓NNNAGG FD1304 36FastDigest® EcoO109I DraII RG↓GNCCY FD0264 37FastDigest® EcoRI EcoRI G↓AATTC FD0274/5 37FastDigest® EcoRV (Eco32I) EcoRV GAT↓ATC FD0303/4 37FastDigest® EheI NarI (GG↓CGCC) GGC↓GCC FD0443/4 38FastDigest® Fnu4HI (SatI) Fnu4HI GC↓NGC FD1644 38FastDigest® FokI FokI GGATG(9/13)↓ FD2144 38FastDigest® FspI (NsbI) MstI TGC↓GCA FD1224 39FastDigest® FspAI FspAI RTGC↓GCAY FD1664 39FastDigest® HaeII (BfoI) HaeII RGCGT↓Y FD2184 39FastDigest® HaeIII (BsuRI) HaeIII GG↓CC FD0154 40FastDigest® HgaI (CseI) HgaI GACGC(5/10)↓ FD1904 40FastDigest® HhaI HhaI GCG↓C FD1854 40FastDigest® HincII HindII GTY↓RAC FD0494 41FastDigest® HindIII HindIII A↓AGCTT FD0504/5 41FastDigest® HinfI HinfI G↓ANTC FD0804 41FastDigest® HinP1I (Hin6I) HhaI (GCG↓C) G↓CGC FD0484 42FastDigest® HpaI (KspAI) HpaI GTT↓AAC FD1034 42FastDigest® HpaII HpaII C↓CGG FD0514 42FastDigest® Hpy8I MjaIV GTN↓NAC FD1574 43FastDigest® HpyF10VI MwoI GCNNNNN↓NNGC FD1734 43
9
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.2. FastDigest® restriction enzymes.
(continued on next page)
FastDigest® restriction enzyme
Prototype Specificity 5’→3’ Cat. # Page
FastDigest® KpnI KpnI GGTAC↓C FD0524 43FastDigest® Kpn2I BspMII T↓CCGGA FD0534 44FastDigest® MauBI MauBI CG↓CGCGCG FD2084 44FastDigest® MboI MboI ↓GATC FD0814 44FastDigest® MboII MboII GAAGA(8/7)↓ FD0824 45FastDigest® MfeI (MunI) MfeI C↓AATTG FD0753/4 45FastDigest® MluI MluI A↓CGCGT FD0564 45FastDigest® MlyI (SchI) PleI (GAGTC(4/5)↓) GAGTC(5/5)↓ FD1374 46FastDigest® MnlI MnlI CCTC(7/6)↓ FD1074 46FastDigest® MreI Sse232I CG↓CCGGCG FD2024 46FastDigest® MscI (MlsI) BalI TGG↓CCA FD1214 47FastDigest® MseI (SaqAI) MseI T↓TAA FD2174 47FastDigest® MslI (RseI) MslI CAYNN↓NNRTG FD2004 47FastDigest® MspI HpaII C↓CGG FD0544 48FastDigest® MssI PmeI GTTT↓AAAC FD1344 48FastDigest® MvaI EcoRII (↓CCWGG) CC↓WGG FD0554 48FastDigest® Mva1269I BsmI GAATGC(1/-1)↓ FD0964 49FastDigest® NaeI (PdiI) NaeI GCC↓GGC FD1524 49FastDigest® NciI (BcnI) CauII CC↓SGG FD0064 49FastDigest® NcoI NcoI C↓CATGG FD0573/4/5 50FastDigest® NdeI NdeI CA↓TATG FD0583/4/5 50FastDigest® NheI NheI G↓CTAGC FD0973/4 50FastDigest® NlaIII (Hin1II) NlaIII CATG↓ FD1834 51FastDigest® NlaIV (BspLI) NlaIV GGN↓NCC FD1154 51FastDigest® NmuCI Tsp45I ↓GTSAC FD1514 51FastDigest® NotI NotI GC↓GGCCGC FD0593/4/6 52FastDigest® NruI (RruI) NruI TCG↓CGA FD2154 52FastDigest® NsiI (Mph1103I) AvaIII ATGCA↓T FD0734 52FastDigest® NspI (XceI) NspI RCATG↓Y FD1474 53FastDigest® PacI PacI TTAAT↓TAA FD2204 53FastDigest® PdmI XmnI GAANN↓NNTTC FD1534 53FastDigest® PflMI (Van91I) PflMI CCANNNN↓NTGG FD0714 54FastDigest® PfoI PfoI T↓CCNGGA FD1754 54FastDigest® PmlI (Eco72I) PmaCI CAC↓GTG FD0364 54FastDigest® PpuMI (Psp5II) PpuMI RG↓GWCCY FD0764 55FastDigest® PshAI (BoxI) PshAI GACNN↓NNGTC FD1434 55FastDigest® PsiI (AanI) PsiI TTA↓TAA FD2064 55FastDigest® PspFI BseYI CCCAGC(-5/-1)↓ FD2224 56FastDigest® PstI PstI CTGCA↓G FD0614/5 56FastDigest® PsuI XhoII R↓GATCY FD1554 56FastDigest® PsyI Tth111I GACN↓NNGTC FD1334 57FastDigest® PvuI PvuI CGAT↓CG FD0624 57FastDigest® PvuII PvuII CAG↓CTG FD0634 57FastDigest® RsaI RsaI GT↓AC FD1124 58FastDigest® RsrII (CpoI) RsrII CG↓GWCCG FD0744 58FastDigest® SacI SacI GAGCT↓C FD1133/4 58FastDigest® SalI SalI G↓TCGAC FD0644 59FastDigest® SanDI (KflI) SanDI GG↓GWCCC FD2164 59FastDigest® SapI (LguI) SapI GCTCTTC(1/4)↓ FD1934 59FastDigest® Sau3AI (Bsp143I) MboI ↓GATC FD0784 60
10
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Alphabetic list of commercially available restriction enzymes see p.207.
Single letter codeR = G or A; H = A, C or T; Y = C or T; V = A, C or G;W = A or T; B = C, G or T;M = A or C; D = A, G or T;K = G or T; N = G, A, T or C.S = C or G;
Table 1.2. FastDigest® restriction enzymes.
FastDigest® restriction enzyme
Prototype Specificity 5’→3’ Cat. # Page
FastDigest® Sau96I (Cfr13I) AsuI G↓GNCC FD0194 60FastDigest® SbfI (SdaI) Sse8387I CCTGCA↓GG FD1194 60FastDigest® ScaI ScaI AGT↓ACT FD0434 61FastDigest® ScrFI (Bme1390I) ScrFI CC↓NGG FD1424 61FastDigest® SexAI (CsiI) SexAI A↓CCWGGT FD2114 61FastDigest® SfaNI (BmsI) SfaNI GCATC(5/9)↓ FD2124 62FastDigest® SfcI (BfmI) SfeI C↓TRYAG FD1164 62FastDigest® SfiI SfiI GGCCNNNN↓NGGCC FD1824 62FastDigest® SmaI SmaI CCC↓GGG FD0663/4 63FastDigest® SnaBI (Eco105I) SnaBI TAC↓GTA FD0404 63FastDigest® SpeI (BcuI) SpeI A↓CTAGT FD1253/4 63FastDigest® SphI (PaeI) SphI GCATG↓C FD0604 64FastDigest® SspI SspI AAT↓ATT FD0774 64FastDigest® StuI (Eco147I) StuI AGG↓CCT FD0424 64FastDigest® StyI (Eco130I) StyI C↓CWWGG FD0414 65FastDigest® SwaI (SmiI) SwaI ATTT↓AAAT FD1244 65FastDigest® TaaI Tsp4CI ACN↓GT FD1364 65FastDigest® TaiI MaeII (A↓CGT) ACGT↓ FD1144 66FastDigest® TaqI TaqI T↓CGA FD0674 66FastDigest® TatI TatI W↓GTACW FD1294 66FastDigest® TauI TauI GCSG↓C FD1654 67FastDigest® TfiI (PfeI) TfiI G↓AWTC FD1784 67FastDigest® Tru1I MseI T↓TAA FD0984 67FastDigest® Tsp509I (TasI) TspEI ↓AATT FD1354 68FastDigest® TspRI (TscAI) TspRI CASTG(2/-7)↓ FD2104 68FastDigest® XapI ApoI R↓AATTY FD1383/4 68FastDigest® XbaI XbaI T↓CTAGA FD0684/5 69FastDigest® XhoI XhoI C↓TCGAG FD0694/5 69
11
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
FastDigest® AccI (XmiI)
Digestion time with 1 µl of FastDigest® AccI (XmiI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 60 min 5 min 2 bp 65°C, 5 min 16 hours
5’...G T↓M K A C...3’
3’...C A K M↑T G...5’
#FD1484 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – blocked (p.178).
λ DN
A, 1
.0%
aga
rose
FastDigest® Acc65I
Digestion time with 1 µl of FastDigest® Acc65I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 65°C, 5 min 16 hours
5’...G↓ G T A C C...3’
3’...C C A T G↑ G...5’
#FD0904 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/BamHI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179).
λ DN
A, 0
.7%
aga
rose
NoteFastDigest® Acc65I cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
FastDigest® AatII
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion time with 1 µl of FastDigest® AatII bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
15 min 20 min 15 min 15 min 5 bp 80°C, 5 min 16 hours
5’...G A C G T↓C...3’
3’...C↑T G C A G...5’
#FD0994 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pUC19 DNA/SmaI fragments in 15 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
pUC1
9 DN
A/Sm
aI, 1
.0%
aga
rose
Product Description
1 cl
eava
ge s
ite9
clea
vage
site
s2
clea
vage
site
s
12
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
FastDigest® AclI (Psp1406I)
Digestion time with 1 µl of FastDigest® AclI (Psp1406I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 65°C, 5 min 16 hours
5’...A A↓C G T T...3’
3’...T T G C↑A A...5’
#FD0944 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not deter-mined.
λ DN
A, 0
.7%
aga
rose
FastDigest® AciI (SsiI)
Digestion time with 1 µl of FastDigest® AciI (SsiI ) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 65°C, 5 min 4 hours
5’...C↓C G C...3’
3’...G G C↑G...5’
#FD1794 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
λ DN
A, 2
.0%
aga
rose
FastDigest® AcuI (Eco57I)
Digestion time with 1 µl of FastDigest® AcuI (Eco57I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
15 min 30 min 15 min 15 min 2 bp 65°C, 5 min 16 hours
5’...C T G A A G (N)16↓...3’
3’...G A C T T C (N)14↑...5’
#FD0344 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml20X SAM (0.2 mM) 20 µl
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 15 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.SAM 0.01 mM.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
λ DN
A, 1
.0%
aga
rose
Note• FastDigest® AcuI (Eco57I) requires only
Mg2+ for its activity, but is stimulated by S-adenosylmethionine. Still, complete cleavage of some substrates with FastDi-gest® AcuI (Eco57I) is difficult to achieve.
• For cleavage with FastDigest® AcuI (Eco57I) at least two copies of its recognition sequence are required.
• FastDigest® AcuI (Eco57I) may remain associated with the cleaved DNA. This may cause DNA band shifting during electropho-resis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.
516
clea
vage
site
s7
clea
vage
site
s40
cle
avag
e si
tes
13
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
FastDigest® AfeI (Eco47III)
Digestion time with 1 µl of FastDigest® AfeI (Eco47III ) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 4 bp 65°C, 5 min 16 hours
5’...A G C↓G C T...3’
3’...T C G↑C G A...5’
#FD0324 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/Eco81I fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: completely overlaps – blocked (p.178).EcoBI: may overlap – effect not determined.
λ DN
A, 0
.7%
aga
rose
FastDigest® AgeI (BshTI)
Digestion time with 1 µl of FastDigest® AgeI (BshTI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 80°C, 5 min 16 hours
5’...A↓C C G G T...3’
3’...T G G C C↑A...5’
#FD1464 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not deter-mined.
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® AflII (BspTI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 4 bp No 16 hours
5’...C↓T T A A G...3’
3’...G A A T T↑C...5’
#FD0834 150 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/BamHI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoBI, EcoKI – no effect.
λ DN
A, 0
.7%
aga
rose
FastDigest® AflII (BspTI)
2 cl
eava
ge s
ites
3 cl
eava
ge s
ites
13 c
leav
age
site
s
14
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® AluI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
15 min 15 min 15 min 15 min 4 bp 65°C, 5 min 16 hours
FastDigest® AluI
5’...A G↓C T...3’
3’...T C↑G A...5’
#FD0014 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 15 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – blocked (p.181).
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® AleI (OliI ) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 10 min 5 min 3 bp 65°C, 5 min 6 hours
5’...C A C N N↓N N G T G...3’
3’...G T G N N↑N N C A C...5’
#FD1634 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of FX174 DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI: may overlap – blocked (p.181).
FastDigest® AleI (OliI)
FX1
74 D
NA, 1
.0%
aga
rose
Note• FastDigest® AjuI requires S-adenosylme-
thionine for activity. Still, complete cleavage of some substrates with FastDigest® AjuI is difficult to achieve.
• FastDigest® AjuI produces double-strand cuts on both sides of the interrupted rec-ognition site. In certain sequence contexts, the cleavage position may be shifted by one base pair. However, the cleavage position indicated above will predominate.
Digestion time with 1 µl of FastDigest® AjuI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 10 min 5 min ND 65°C, 5 min 16 hours
5’...↓ 7(N) G A A(N)7 T T G G (N)11↓...3’
3’...↑ 12(N) C T T(N)7 A A C C (N)6↑ ...5’
#FD1954 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml20X SAM (0.2 mM) 20 µl
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of FX174 DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.SAM 0.01 mM.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
FX1
74 D
NA, 1
.0%
aga
rose
FastDigest® AjuI
1 cl
eava
ge s
ite1
clea
vage
site
143
clea
vage
site
s
15
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
FastDigest® AlwNI (CaiI)
Digestion time with 1 µl of FastDigest® AlwNI (CaiI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 4 bp 65°C, 10 min 16 hours
5’...C A G N N N↓C T G...3’
3’...G T C↑N N N G A C...5’
#FD1394 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, CpG, EcoKI – no effect.Dcm: may overlap – blocked (p.177).EcoBI: may overlap – effect not determined.
λ DN
A, 0
.7%
aga
rose Note
FastDigest® AlwNI (CaiI) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Digestion time with 1 µl of FastDigest® Alw21I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp 80°C, 20 min 16 hours
5’...G W G C W↓C...3’
3’...C↑W C G W G...5’
#FD0024 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
λ DN
A, 1
.0%
aga
rose
FastDigest® Alw21I
28 c
leav
age
site
s
FastDigest® Alw26I
Digestion time with 1 µl of FastDigest® Alw26I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp 65°C, 5 min 16 hours
5’...G T C T C(N)1↓...3’
3’...C A G A G(N)5↑...5’
#FD0034 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
λ DN
A, 1
.0%
aga
rose
37 c
leav
age
site
s41
cle
avag
e si
tes
16
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
FastDigest® ApaLI (Alw44I)
Digestion time with 1 µl of FastDigest® ApaLI (Alw44I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 60 min 10 min 5 min 4 bp 80°C, 5 min 16 hours
5’...G↓T G C A C...3’
3’...C A C G T↑G...5’
#FD0044 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/SmaI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – blocked (p.179).
λ DN
A, 0
.7%
aga
rose
FastDigest® AscI (SgsI)
Digestion time with 1 µl of FastDigest® AscI (SgsI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 65°C, 20 min 16 hours
5’...G G↓C G C G C C...3’
3’...C C G C G C↑G G...5’
#FD1894 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/CpoI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
λ DN
A, 0
.7%
aga
rose
FastDigest® ApaI
Digestion time with 1 µl of FastDigest® ApaI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 20 min 10 min 2 bp 65°C, 5 min 16 hours
5’...G G G C C↓C...3’
3’...C↑C C G G G...5’
#FD1414 300 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/CpoI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179).
λ DN
A, 0
.7%
aga
rose
NoteFastDigest® ApaI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).1
clea
vage
site
4 cl
eava
ge s
ites
2 cl
eava
ge s
ites
17
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
FastDigest® AseI (VspI)
Digestion time with 1 µl of FastDigest® AseI (VspI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 30 min 2 bp 65°C, 5 min 16 hours
5’...A T↓T A A T...3’
3’...T A A T↑T A...5’
#FD0914 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
λ DN
A , 1
.0%
aga
rose
FastDigest® AvaI (Eco88I)
Digestion time with 1 µl of FastDigest® AvaI (Eco88I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 65°C, 5 min 16 hours
5’...C↓Y C G R G...3’
3’...G R G C Y↑C...5’
#FD0384 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – cleavage impaired (p.178).
λ DN
A, 0
.7%
aga
rose
FastDigest® AsiSI (SfaAI)
Digestion time with 1 µl of FastDigest® AsiSI (SfaAI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
no cleavage sites 5 min 60 min 5 min 5 bp 80°C, 5 min 6 hours
5’...G C G A T↓ C G C...3’
3’...C G C↑ T A G C G...5’
#FD2094 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of linearized pJET1 DNA with inserted SfaAI recognition site in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
pJET
1-Sf
aAI D
NA/S
daI,
1.0%
aga
rose
17 c
leav
age
site
s1
clea
vage
site
8 cl
eava
ge s
ites
18
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
FastDigest® AvrII (XmaJI)
Digestion time with 1 µl of FastDigest® AvrII (XmaJI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 10 min 5 min 10 min 2 bp No 16 hours
5’...C↓C T A G G...3’
3’...G G A T C↑C...5’
#FD1564 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/SmaI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
λ DN
A, 1
.0%
aga
rose
FastDigest® BamHI
Digestion time with 1 µl of FastDigest® BamHI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 80°C, 5 min 1 hour
5’...G↓G A T C C...3’
3’...C C T A G↑G...5’
#FD0054 800 µlSupplied with:10X FastDigest® buffer 2x1 ml10X FastDigest® Green Buffer 2x1 ml
#FD0055 2500 µlSupplied with:10X FastDigest® buffer 5x1 ml10X FastDigest® Green Buffer 5x1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/Bsp120I fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
λ DN
A, 0
.7%
aga
rose
FastDigest® AvaII (Eco47I)
Digestion time with 1 µl of FastDigest® AvaII (Eco47I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp 80°C, 20 min 16 hours
5’...G↓G W C C ...3’
3’...C C W G↑G...5’
#FD0314 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – blocked (p.177, 179).
λ DN
A, 1
.0%
aga
rose
NoteFastDigest® AvaII (Eco47I) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
35 c
leav
age
site
s2
clea
vage
site
s5
clea
vage
site
s
19
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
FastDigest® BanI (BshNI)
Digestion time with 1 µl of FastDigest® BanI (BshNI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 60 min 5 min 15 min 2 bp 65°C, 10 min 16 hours
5’...G↓G Y R C C...3’
3’...C C R Y G↑G...5’
#FD1004 300 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA dcm– in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, EcoBI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179).EcoKI: may overlap – effect not determined.
λ DN
A (d
cm– ),
1.0
% a
garo
se
NoteFastDigest® BanI (BshNI) cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Digestion time with 1 µl of FastDigest® BbvI (Lsp1109I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 10 min 2 bp 65°C, 5 min 1 hour
5’...G C A G C (N)8 ↓...3’
3’...C G T C G (N)12↑...5’
#FD2074 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pBR322 DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
pBR3
22 D
NA, 0
.7%
aga
rose
FastDigest® BbvI (Lsp1109I)
FastDigest® BbsI (BpiI)
Digestion time with 1 µl of FastDigest® BbsI (BpiI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp 65°C, 10 min 16 hours
5’...G A A G A C(N)2↓...3’
3’...C T T C T G(N)6↑...5’
#FD1014 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
λ DN
A. 0
.7%
aga
rose
25 c
leav
age
site
s24
cle
avag
e si
tes
21 c
leav
age
site
s
20
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
FastDigest® BglI
Digestion time with 1 µl of FastDigest® BglI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 65°C, 5 min 2 hours
5’...G C C N N N N↓N G G C...3’
3’...C G G N↑N N N N C C G...5’
#FD0074 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
λ DN
A, 0
.7%
aga
rose
FastDigest® BclI
Digestion time with 1 µl of FastDigest® BclI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 15 min 5 min 3 bp 80°C, 20 min 16 hours
5’...T↓G A T C A...3’
3’...A C T A G↑T...5’
#FD0724 300 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA dam– in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam: completely overlaps – blocked (p.176).Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – blocked (p.181).
λ DN
A (d
am–),
0.7%
aga
rose
NoteFastDigest® BclI is blocked by dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
FastDigest® BfaI (FspBI)
Digestion time with 1 µl of FastDigest® BfaI (FspBI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 15 min 5 min 20 min 3 bp 80°C, 5 min 16 hours
5’...C↓T A G...3’
3’...G A T↑C...5’
#FD1764 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
λ DN
A, 0
.7%
aga
rose
8 cl
eava
ge s
ites
13 c
leav
age
site
s29
cle
avag
e si
tes
21
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
FastDigest® BglII
Digestion time with 1 µl of FastDigest® BglII bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 20 min 30 min 20 min 3 bp No 16 hours
5’...A↓G A T C T...3’
3’...T C T A G↑A...5’
#FD0083 100 µl #FD0084 200 µl
Both supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – cleavage impaired (p.181).
λ DN
A, 0
.7%
aga
rose
FastDigest® BlpI (Bpu1102I)
Digestion time with 1 µl of FastDigest® BlpI (Bpu1102I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 80°C, 5 min 16 hours
5’...G C↓T N A G C...3’
3’...C G A N T↑C G...5’
#FD0094 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
λ DN
A, 0
.7%
aga
rose
FastDigest® Bme1580I (BseSI)
Digestion time with 1 µl of FastDigest® Bme1580I (BseSI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 30 min 3 bp 80°C, 15 min 16 hours
5’...G K G C M↓C...3’
3’...C↑M C G K G...5’
#FD1444 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – cleavage impaired (p.181).
λ DN
A, 0
.7%
aga
rose
6 cl
eava
ge s
ites
6 cl
eava
ge s
ites
10 c
leav
age
site
s
22
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
FastDigest® BpmI (GsuI)
Digestion time with 1 µl of FastDigest® BpmI (GsuI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
15 min 30 min 15 min 15 min 2 bp 65°C, 5 min 16 hours
5’...C T G G A G (N)16↓...3’
3’...G A C C T C (N)14↑...5’
#FD0464 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 15 min at 30°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 30°C.
Methylation EffectsDam, CpG, EcoKI, EcoBI – no effect.Dcm: may overlap – blocked (p.177).
λ DN
A, 1
.0%
aga
rose
Note• FastDigest® BpmI (GsuI) requires only
Mg2+ for its activity, but is stimulated by S-adenosylmethionine.
• FastDigest® BpmI (GsuI) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
FastDigest® BplI
Digestion time with 1 µl of FastDigest® BplI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
15 min 20 min 30 min 30 min ND 65°C, 5 min 16 hours
5’...↓ 8(N) G A G(N)5 C T C(N)13↓...3’
3’...↑ 13(N) C T C(N)5 G A G(N)8↑ ...5’
#FD1314 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml20X SAM (1.0 mM) 20 µl
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/XhoI fragments in 15 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.SAM 0.05 mM.
λ DN
A/Xh
oI, 0
.7%
aga
rose Note
FastDigest® BplI requires only Mg2+ for its acti-vity, but is stimulated by S-adenosylmethionine. Still, complete cleavage of some substrates with FastDigest® BplI is difficult to achieve.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion time with 1 µl of FastDigest® BmtI (BspOI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 15 min 5 min 3 bp 80°C, 5 min 1 hour
5’...G C T A G↓C...3’
3’...C↑G A T C G...5’
#FD2044 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/HindIII fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
λ DN
A, 0
.7%
aga
rose
FastDigest® BmtI (BspOI)
1 cl
eava
ge s
ite1
clea
vage
site
25 c
leav
age
site
s
23
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
FastDigest® BsaBI (BseJI)
Digestion time with 1 µl of FastDigest® BsaBI (BseJI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp No 1 hour
5’...G A T N N↓N N A T C...3’
3’...C T A N N↑N N T A G...5’
#FD1714 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA dam– in 5 min at 65°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 65°C.
Methylation EffectsDam: may overlap – blocked (p.176).Dcm, EcoKI, CpG – no effect.EcoBI: may overlap – effect not determined.
λ DN
A (d
am–),
1.0%
aga
rose
NoteFastDigest® BsaBI (BseJI) is blocked by overlapping dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
FastDigest® BsaAI (Ppu21I)
Digestion time with 1 µl of FastDigest® BsaAI (Ppu21I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp 65°C, 5 min 6 hours
5’...Y A C↓G T R...3’
3’...R T G↑C A Y...5’
#FD1974 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not determined.
λ DN
A, 0
.7%
aga
rose
FastDigest® Bpu10I
Digestion time with 1 µl of FastDigest® Bpu10I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
15 min 15 min 30 min 15 min 3 bp 80°C, 5 min 1 hour
5’...C C↓T N A G C...3’
3’...G G A N T↑C G...5’
#FD1184 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of M13mp18 DNA in 15 min at 37°C in 1X Fast-Digest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
M13
mp1
8 DN
A, 0
.7%
aga
rose
NoteFor cleavage with FastDigest® Bpu10I at least two copies of its recognition sequence are required.
4 cl
eava
ge s
ites
14 c
leav
age
site
s21
cle
avag
e si
tes
24
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
FastDigest® BsaJI (BseDI)
Digestion time with 1 µl of FastDigest® BsaJI (BseDI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 80°C, 5 min 16 hours
5’...C↓C N N G G...3’
3’...G G N N C↑C...5’
#FD1084 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® BseGI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 80°C, 5 min 16 hours
5’...G G A T G N N↓...3’
3’...C C T A C↑N N ...5’
#FD0874 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® BseGI
λ DN
A, 1
.0%
aga
rose
FastDigest® BsaHI (Hin1I)
Digestion time with 1 µl of FastDigest® BsaHI (Hin1I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp 65°C, 5 min 16 hours
5’...G R↓C G Y C...3’
3’...C Y G C↑R G...5’
#FD0474 100 µl Supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA dcm– in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, EcoKI – no effect.Dcm: may overlap – cleavage impaired (p.177).CpG: completely overlaps – blocked (p.178).EcoBI: may overlap – effect not determined.
λ DN
A (d
cm– ),
1.0
% a
garo
se
NoteFastDigest® BsaHI (Hin1I) cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
40 c
leav
age
site
s10
5 cl
eava
ge s
ites
150
clea
vage
site
s
25
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
FastDigest® Bsh1236I
Digestion time with 1 µl of FastDigest® Bsh1236I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp 80°C, 10 min 16 hours
5’...C G↓C G...3’
3’...G C↑G C...5’
#FD0924 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
λ DN
A, 1
.4%
aga
rose
FastDigest® BseNI
Digestion time with 1 µl of FastDigest® BseNI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 80°C, 5 min 16 hours
5’...A C T G G N↓...3’
3’...T G A C↑C N ...5’
#FD0884 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 65°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 65°C.
Methylation EffectsDam, Dcm, CpG – no effect.EcoKI, EcoBI: may overlap – effect not deter-mined.
λ DN
A, 1
.0%
aga
rose
FastDigest® BseXI
Digestion time with 1 µl of FastDigest® BseXI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
15 min 15 min 15 min 15 min 3 bp 80°C, 15 min 16 hours
5’...G C A G C(N)8↓ ...3’
3’...C G T C G(N)12↑...5’
#FD1454 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pBR322 DNA in 15 min at 65°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 65°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
pBR3
22 D
NA, 1
.4%
aga
rose
110
clea
vage
site
s21
cle
avag
e si
tes
157
clea
vage
site
s
26
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
FastDigest® BsiWI (Pfl23II)
Digestion time with 1 µl of FastDigest® BsiWI (Pfl23II) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
15 min 15 min 15 min 15 min 3 bp 65°C, 5 min 16 hours
5’...C↓G T A C G...3’
3’...G C A T G↑C...5’
#FD0854 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/Psp1406I fragments in 15 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsCpG: completely overlaps – blocked (p.178).Dam, Dcm, EcoKI, EcoBI – no effect.
λ DN
A, 0
.7%
aga
rose
FastDigest® BslI (BseLI)
Digestion time with 1 µl of FastDigest® BslI (BseLI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp No 16 hours
5’...C C N N N N N↓N N G G...3’
3’...G G N N↑N N N N N C C...5’
#FD1204 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA dcm– in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179).
λ DN
A (d
cm– ),
1.4
% a
garo
se
NoteFastDigest® BslI (BseLI) cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
FastDigest® BsiEI (Bsh1285I)
Digestion time with 1 µl of FastDigest® BsiEI (Bsh1285I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
15 min 15 min 15 min 15 min 3 bp 80°C, 15 min 1 hour
5’...C G R Y↓C G...3’
3’...G C↑Y R G C...5’
#FD0894 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 15 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam: may overlap – cleavage impaired (p.176).Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
λ DN
A, 1
.0%
aga
rose
NoteFastDigest® BsiEI (Bsh1285I) cleavage is impaired by overlapping dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).22
cle
avag
e si
tes
1 cl
eava
ge s
ite17
6 cl
eava
ge s
ites
27
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® Bsp119I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp 80°C, 5 min 16 hours
5’...T T↓C G A A...3’
3’...A A G C↑T T...5’
#FD0124 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).EcoKI: may overlap – effect not determined.
FastDigest® Bsp119I
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® BsmFI (FaqI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
15 min 15 min 15 min 15 min 3 bp 65°C, 5 min 16 hours
5’...G G G A C(N)10↓...3’
3’...C C C T G(N)14↑...5’
#FD1814 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml20X SAM (1.0 mM) 20 µl
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 15 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.SAM 0.05 mM.
FastDigest® BsmFI (FaqI)
λ DN
A, 1
.0%
aga
rose
Note• FastDigest® BsmFI (FaqI) requires only Mg2+
for its activity, but is stimulated by S-ade-nosylmethionine. Still, complete cleavage of some substrates is difficult to achieve.
• For cleavage with FastDigest® BsmFI (FaqI) at least two copies of its recognition sequence are required.
Methylation EffectsDam, Dcm, EcoBI, EcoKI – no effect.CpG: may overlap – blocked (p.179).
FastDigest® BsmBI (Esp3I)
Digestion time with 1 µl of FastDigest® BsmBI (Esp3I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp 65°C, 10 min 6 hours
5’...C G T C T C(N)1↓...3’
3’...G C A G A G(N)5↑...5’
#FD0454 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.DTT (#R0861/2) 1 mM.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: completely overlaps – blocked (p.178).EcoBI: may overlap – effect not determined.
λ DN
A, 1
.0%
aga
rose Note
The enzyme requires DTT (#R0861/2). Freshly made DTT should be added to the reaction buffer.
14 c
lava
ge s
ites
38 c
leav
age
site
s7
clea
vage
site
s
28
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® Bsp1407I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 80°C, 10 min 16 hours
5’...T↓G T A C A...3’
3’...A C A T G↑T...5’
#FD0933 50 µl#FD0934 100 µl
Both supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
FastDigest® Bsp1407I
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® Bsp120I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 15 min 5 min 3 bp 80°C, 10 min 16 hours
5’...G↓G G C C C...3’
3’...C C C G G↑G...5’
#FD0134 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/CpoI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – blocked (p.177, 179).
FastDigest® Bsp120I
λ DN
A, 0
.7%
aga
rose
NoteFastDigest® Bsp120I is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Digestion time with 1 µl of FastDigest® Bsp1286I (SduI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 80°C, 15 min 1 hour
5’...G D G C H↓C...3’
3’...C↑H C G D G...5’
#FD0654 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG – no effect.EcoKI, EcoBI: may overlap – effect not determined.
λ DN
A, 1
.0%
aga
rose
FastDigest® Bsp1286I (SduI)
1 cl
eava
ge s
ite38
cle
avag
e si
tes
5 cl
eava
ge s
ites
29
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® BspCNI (BseMII) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
15 min 30 min 15 min 15 min 2 bp 80°C, 5 min 16 hours
5’...C T C A G(N)10↓...3’
3’...G A G T C(N)8↑ ...5’
#FD1404 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml20X SAM (0.2 mM) 20 µl
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 15 min at 55°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 55°C.SAM 0.01 mM.
Methylation EffectsDam, Dcm, CpG, EcoBI, EcoKI – no effect.
FastDigest® BspCNI (BseMII)
λ DN
A, 1
.0%
aga
rose
NoteFastDigest® BspCNI (BseMII) requires S-adenosylmethionine for activity. Sinefungin can replace SAM in the restriction reaction. In this case DNA is not methylated and more than 95% of the ligated BspCNI (BseMII) fragments can be recut by this enzyme.
Digestion time with 1 µl of FastDigest® BspHI (PagI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 10 min 5 min 10 min 3 bp 80°C, 5 min 0.5 hour
5’...T↓C A T G A...3’
3’...A G T A C↑T...5’
#FD1284 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of FX174 DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDcm, CpG, EcoKI – no effect.Dam, EcoBI: may overlap – cleavage impaired (p.176, 181).
FastDigest® BspHI (PagI)
FX1
74 D
NA, 1
.0%
aga
rose
NoteFastDigest® BspHI (PagI) cleavage is impaired by overlapping dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Digestion time with 1 µl of FastDigest® BspMI (BveI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
15 min 15 min 60 min 20 min 5 bp 65°C, 5 min 16 hours
5’...A C C T G C(N)4↓...3’
3’...T G G A C G(N)8↑...5’
#FD1744 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml20X Oligonucleotide (0.01 mM) 20 µl
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 15 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.Oligonucleotide 0.5 µM.
Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI, EcoKI: may overlap – effect not determined.
FastDigest® BspMI (BveI)
λ DN
A, 0
.7%
aga
rose
sequence in the reaction mixture signifi-cantly improves cleavage of plasmid DNAs, especially of those with a single FastDi-gest® BspMI (BveI) site. Still, complete cleavage of some substrates is difficult to achieve.
Note• At least two copies of FastDigest® BspMI
(BveI) recognition site are required for efficient cleavage.
• Inclusion of 0.5 µM oligonucleotide with the FastDigest® BspMI (BveI) recognition
• FastDigest® BspMI (BveI) may remain associated with the cleaved DNA. This may cause DNA band shifting during electropho-resis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.
79 c
leav
age
site
s3
clea
vage
site
s41
cle
avag
e si
tes
30
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® BsrDI (BseMI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
15 min 15 min 15 min 15 min 3 bp 80°C, 5 min 16 hours
5’...G C A A T G N N↓...3’
3’...C G T T A C↑N N ...5’
#FD1264 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 15 min at 55°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 55°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® BsrDI (BseMI)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® BsrBI (MbiI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 65°C, 5 min 16 hours
5’...C C G↓C T C...3’
3’...G G C↑G A G...5’
#FD1274 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: completely overlaps – cleavage impaired (p.178).EcoBI: may overlap – effect not determined.
FastDigest® BsrBI (MbiI)
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® BsrFI (Cfr10I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 10 min 5 min 2 bp No 2 hours
5’...R↓C C G G Y...3’
3’...Y G G C C↑R...5’
#FD0184 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not determined.
FastDigest® BsrFI (Cfr10I)
λ DN
A, 1
.0%
aga
rose
NoteFor cleavage with FastDigest® BsrFI (Cfr10I) at least two copies of its recognition sequence are required.
17 c
leav
age
site
s44
cle
avag
e si
tes
61 c
leav
age
site
s
31
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® BstZ17I (Bst1107I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 10 min 3 bp No 6 hours
5’...G T A↓T A C...3’
3’...C A T↑A T G...5’
#FD0704 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
FastDigest® BstZ17I (Bst1107I)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® BstXI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 4 bp 80°C, 5 min 0.5 hours
5’...C C A N N N N N↓N T G G...3’
3’...G G T N↑N N N N N A C C...5’
#FD1024 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, CpG, EcoKI, EcoBI – no effect.Dcm: may overlap – cleavage impaired (p.177).
FastDigest® BstXI
λ DN
A, 0
.7%
aga
rose
NoteFastDigest® BstXI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Digestion time with 1 µl of FastDigest® BssHII (PteI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 80°C, 5 min 16 hours
5’...G↓C G C G C...3’
3’...C G C G C↑G...5’
#FD2134 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® BssHII (PteI)
λ DN
A, 0
.7%
aga
rose
6 cl
eava
ge s
ites
13 c
leav
age
site
s3
clea
vage
site
s
32
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® Csp6I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 80°C, 10 min 16 hours
5’...G↓T A C...3’
3’...C A T↑G...5’
#FD0214 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
FastDigest® Csp6I
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® Bsu36I (Eco81I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 80°C, 10 min 16 hours
5’...C C↓T N A G G...3’
3’...G G A N T↑C C...5’
#FD0374 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/HindIII fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® Bsu36I (Eco81I)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® ClaI (Bsu15I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 65°C, 15 min 16 hours
5’...A T↓C G A T...3’
3’...T A G C↑T A...5’
#FD0143 50 µl#FD0144 100 µl
Both supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam: may overlap – blocked (p.176).CpG: completely overlaps – blocked (p.178).Dcm, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® ClaI (Bsu15I)
λ DN
A, 0
.7%
aga
rose
NoteFastDigest® ClaI (Bsu15I) is blocked by overlapping dam methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
2 cl
eava
ge s
ites
15 c
leav
age
site
s11
3 cl
eava
ge s
ites
33
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® DpnI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
not determined 5 min not applicable 10 min 1 bp 80°C, 5 min 16 hours
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pBR322 DNA (dam methylated) in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam: does not cut dam– DNA (p.176).Dcm, EcoKI, CpG – no effect.EcoBI: may overlap – effect not determined.
FastDigest® DpnI
pBR3
22 D
NA, 1
.4%
aga
rose
Note• FastDigest® DpnI requires the presence
of N6-methyladenine within the recognition sequence to cleave DNA.
• DNA purified from a dam+ strain will be a substrate for FastDigest® DpnI.
• FastDigest® DpnI will only cleave fully-adenomethylated dam sites.
• FastDigest® DpnI, FastDigest® Sau3AI (Bsp143I) and FastDigest® MboI all recog-nize the same sequence but have different methylation sensitivities (pp.176-181) and cleavage positions.
5’...G m6A↓ T C...3’
3’...C T↑m6A G...5’
#FD1703 50 µl #FD1704 100 µl
Both supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Digestion time with 1 µl of FastDigest® DdeI (HpyF3I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 10 min 3 bp 65°C, 5 min 16 hours
5’...C↓T N A G...3’
3’...G A N T↑C...5’
#FD1884 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® DdeI (HpyF3I)
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® DraI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 4 bp 65°C, 5 min 16 hours
5’...T T T↓A A A...3’
3’...A A A↑T T T...5’
#FD0224 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – blocked (p.181).
FastDigest® DraI
λ DN
A, 0
.7%
aga
rose
104
clea
vage
site
s22
cle
avag
e si
tes
13 c
leav
age
site
s
34
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® EagI (Eco52I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 20 min 20 min 5 min 3 bp 65°C, 5 min 16 hours
5’...C↓G G C C G...3’
3’...G C C G G↑C...5’
#FD0334 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/Eco81I fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® EagI (Eco52I)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® DraIII (AdeI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 80°C, 5 min 6 hours
5’...C A C N N N↓G T G...3’
3’...G T G↑N N N C A C...5’
#FD1234 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI: may overlap – effect not determined.
FastDigest® DraIII (AdeI)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® DrdI (AasI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp 80°C, 10 min 1 hour
5’...G A C N N N N↓N N G T C...3’
3’...C T G N N↑N N N N C A G...5’
#FD1724 25 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoBI, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).
λ DN
A, 0
.7%
aga
rose
FastDigest® DrdI (AasI)
10 c
leav
age
site
s3
clea
vage
site
s2
clea
vage
site
s
35
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® Ecl136II bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp 65°C, 5 min 6 hours
5’...G A G↓C T C...3’
3’...C T C↑G A G...5’
#FD0254 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/HindIII fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – effect not determined.
FastDigest® Ecl136II
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® EarI (Eam1104I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 80°C, 5 min 6 hours
5’...C T C T T C(N)1↓...3’
3’...G A G A A G(N)4↑...5’
#FD0234 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of FX174 DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® EarI (Eam1104I)
FX1
74 D
NA, 1
.0%
aga
rose
NoteCertain sites in λ DNA are difficult to cleave with FastDigest® EarI (Eam1104I), the same as with its prototype Ksp632I.
Digestion time with 1 µl of FastDigest® Eam1105I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 65°C, 5 min 16 hours
5’...G A C N N N↓N N G T C...3’
3’...C T G N N↑N N N C A G...5’
#FD0244 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
FastDigest® Eam1105I
λ DN
A, 0
.7%
aga
rose
9 cl
eava
ge s
ites
2 cl
eava
ge s
ites
2 cl
eava
ge s
ites
36
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® Eco91I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 65°C, 10 min 2 hours
5’...G↓G T N A C C...3’
3’...C C A N T G↑G...5’
#FD0394 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG – no effect.EcoKI, EcoBI: may overlap – effect not determined.
FastDigest® Eco91I
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® EcoNI (XagI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 20 min 2 bp 65°C, 5 min 16 hours
5’...C C T N N↓N N N A G G...3’
3’...G G A N N N↑N N T C C...5’
#FD1304 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® EcoNI (XagI)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® Eco31I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 65°C, 5 min 16 hours
5’...G G T C T C(N)1↓...3’
3’...C C A G A G(N)5↑...5’
#FD0293 50 µl #FD0294 100 µl
Both supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA dcm–/HindIII fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, EcoKI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179).EcoBI: may overlap – effect not determined.
FastDigest® Eco31I
λ DN
A (d
cm–),
0.7%
aga
rose
NoteFastDigest® Eco31I cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
2 cl
eava
ge s
ites
13 c
leav
age
site
s9
clea
vage
site
s
37
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® EcoRV (Eco32I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp No 16 hours
5’...G A T↓A T C...3’
3’...C T A↑T A G...5’
#FD0303 200 µl #FD0304 400 µl
Both supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® EcoRV (Eco32I)
λ DN
A, 1
.0%
aga
rose
Digestion time with 1 µl of FastDigest® EcoO109I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 10 min 15 min 5 min 2 bp 65°C, 5 min 16 hours
5’...R G↓G N C C Y...3’
3’...Y C C N G↑G R...5’
#FD0264 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/CpoI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, CpG, EcoKI – no effect.Dcm: may overlap – blocked (p.177).EcoBI: may overlap – effect not determined.
FastDigest® EcoO109I
λ DN
A, 0
.7%
aga
rose Note
FastDigest® EcoO109I is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Digestion time with 1 µl of FastDigest® EcoRI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 20 min 5 min 2 bp 80°C, 5 min 0.5 hour
5’...G↓A A T T C...3’
3’...C T T A A↑G...5’
#FD0274 800 µlSupplied with:10X FastDigest® buffer 2x1 ml10X FastDigest® Green Buffer 2x1 ml
#FD0275 2500 µlSupplied with:10X FastDigest® buffer 5x1 ml10X FastDigest® Green Buffer 5x1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
FastDigest® EcoRI
λ DN
A, 0
.7%
aga
rose
3 cl
evag
e si
tes
5 cl
eava
ge s
ites
21 c
leav
age
site
s
38
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® EheI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 65°C, 5 min 6 hours
5’...G G C↓G C C...3’
3’...C C G↑C G G...5’
#FD0443 20 µl #FD0444 50 µl
Both supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/PstI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® EheI
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® FokI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp 65°C, 5 min 1 hour
5’...G G A T G(N)9↓...3’
3’...C C T A C(N)13↑...5’
#FD2144 100 µlBoth supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® FokI
λ DN
A, 1
.0%
aga
rose
Note• At least two copies of FastDigest® FokI
recognition site are required for an efficient cleavage.
• FastDigest® FokI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.
Digestion time with 1 µl of FastDigest® Fnu4HI (SatI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 10 min 5 min 3 bp 65°C, 5 min 16 hours
5’...G C↓N G C...3’
3’...C G N↑C G...5’
#FD1644 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – blocked (p.179).
FastDigest® Fnu4HI (SatI)
λ DN
A, 1
.4%
aga
rose
NoteAt least two copies of FastDigest® Fnu4HI (SatI) recognition site are required for an efficient cleavage.
1 cl
eava
ge s
ite38
0 cl
eava
ge s
ites
150
clea
vage
site
s
39
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® FspI (NsbI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 65°C, 15 min 16 hours
5’...T G C↓G C A...3’
3’...A C G↑C G T...5’
#FD1224 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® FspI (NsbI)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® FspAI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 4 bp 65°C, 5 min 16 hours
5’...R T G C↓G C A Y...3’
3’...Y A C G↑C G T R...5’
#FD1664 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/Psp1406I fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not determined.
FastDigest® FspAI
λ DN
A, 0
.5%
aga
rose
5’...R G C G C↓Y...3’
3’...Y↑C G C G R...5’
#FD2184 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® HaeII (BfoI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 65°C, 10 min 1 hour
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: completely overlaps – blocked (p.178).EcoBI: may overtlap – not determined.
FastDigest® HaeII (BfoI)
15 c
leav
age
site
s2
clea
vage
site
s48
cle
avag
e si
tes
40
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® HhaI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp No 16 hours
5’...G C G↓C...3’
3’...C↑G C G...5’
#FD1854 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® HhaI
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® HaeIII (BsuRI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp No 16 hours
5’...G G↓C C...3’
3’...C C↑G G...5’
#FD0154 400 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
FastDigest® HaeIII (BsuRI)
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® HgaI (CseI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
15 min 15 min 30 min 15 min 2 bp 80°C, 5 min 16 hours
5’...G A C G C(N)5↓...3’
3’...C T G C G(N)10↑...5’
#FD1904 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pBR322 DNA in 15 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI: never overlaps – no effect.CpG: completely overlaps – blocked (p.178).EcoBI: may overlap – effect not determined.
FastDigest® HgaI (CseI)
pBR3
22 D
NA, 1
.4%
aga
rose
Note• For cleavage with FastDigest® HgaI (CseI) at
least two copies of its recognition sequence are required.
• FastDigest® HgaI (CseI) may remain associ-ated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.
149
clea
vage
site
s11
cle
avag
e si
tes
215
clea
vage
site
s
41
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® HincII bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 10 min 1 bp 65°C, 5 min 16 hours
5’...G T Y↓R A C...3’
3’...C A R↑Y T G...5’
#FD0494 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI: may overlap – blocked (p.181).
FastDigest® HincII
λ DN
A, 1
.0%
aga
rose
Digestion time with 1 µl of FastDigest® HindIII bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 20 min 10 min 3 bp 80°C, 10 min 16 hours
5’...A↓A G C T T...3’
3’...T T C G A↑A...5’
#FD0504 800 µlSupplied with:10X FastDigest® buffer 2x1 ml10X FastDigest® Green Buffer 2x1 ml
#FD0505 2500 µlSupplied with:10X FastDigest® buffer 5x1 ml10X FastDigest® Green Buffer 5x1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – cleavage impaired (p.181).
FastDigest® HindIII
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® HinfI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 65°C, 20 min 16 hours
5’...G↓A N T C...3’
3’...C T N A↑G...5’
#FD0804 400 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – blocked (p.181).
FastDigest® HinfI
λ DN
A, 1
.4%
aga
rose
35 c
leav
age
site
s7
clea
vage
site
s14
8 cl
eava
ge s
ites
42
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® HinP1I (Hin6I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 10 min 5 min 5 min 2 bp 80°C, 10 min 16 hours
5’...G↓C G C...3’
3’...C G C↑G...5’
#FD0484 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® HinP1I (Hin6I)
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® HpaII bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 65°C, 5 min 16 hours
5’...C↓C G G...3’
3’...G G C↑C...5’
#FD0514 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® HpaII
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® HpaI (KspAI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 65°C, 20 min 0.5 hour
5’...G T T↓A A C...3’
3’...C A A↑T T G...5’
#FD1034 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI: may overlap – blocked (p.181).
FastDigest® HpaI (KspAI)
λ DN
A, 0
.7%
aga
rose
215
clea
vage
site
s14
cle
avag
e si
tes
328
clea
vage
site
s
43
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® Hpy8I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 10 min 2 bp 80°C, 5 min 16 hours
5’...G T N↓N A C...3’
3’...C A N↑N T G...5’
#FD1574 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI – effect not determined.
FastDigest® Hpy8I
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® HpyF10VI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 80°C, 5 min 16 hours
5’...G C N N N N N↓N N G C...3’
3’...C G N N↑N N N N N C G...5’
#FD1734 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
FastDigest® HpyF10VI
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® KpnI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 80°C, 5 min 16 hours
5’...G G T A C↓C...3’
3’...C↑C A T G G...5’
#FD0524 300 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/BamHI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoKI, EcoBI – no effect.
FastDigest® KpnI
λ DN
A, 0
.7%
aga
rose
125
clea
vage
site
s34
6 cl
eava
ge s
ites
2 cl
eava
ge s
ites
44
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® Kpn2I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 80°C, 5 min 16 hours
5’...T↓C C G G A...3’
3’...A G G C C↑T...5’
#FD0534 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® Kpn2I
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® MauBI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
no cleavage sites 5 min 10 min 5 min 5 bp 65°C, 5 min 16 hours
5’...C G↓C G C G C G...3’
3’...G C G C G C↑G C...5’
#FD2084 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of linear-ized pJET1 DNA with inserted MauBI recognition sites in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® MauBI
pJET
1-M
auBI
DNA
/Bam
HI, 0
.7%
aga
rose
Note• FastDigest® MboI is blocked by overlapping
dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
• FastDigest® MboI, FastDigest® Sau3AI (Bsp143I) and FastDigest® DpnI all recog-nize the same sequence but have different methylation sensitivities (pp.176-181) and cleavage positions.
Digestion time with 1 µl of FastDigest® MboI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 10 min 1 bp 65°C, 15 min 16 hours
5’...↓G A T C ...3’
3’... C T A G↑...5’
#FD0814 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA dam– in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam: completely overlaps – blocked (p.176).EcoBI: may overlap – blocked (p.181).Dcm, CpG, EcoKI – no effect.
FastDigest® MboI
λ DN
A (d
am–),
1.4%
aga
rose
24 c
leav
age
site
s2
cleav
age
sites
116
clea
vage
site
s
45
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® MfeI (MunI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp No 16 hours
5’...C↓A A T T G...3’
3’...G T T A A↑C...5’
#FD0753 20 µl #FD0754 50 µl
Both supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® MfeI (MunI)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® MluI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 15 min 3 bp 80°C, 5 min 16 hours
5’...A↓C G C G T...3’
3’...T G C G C↑A...5’
#FD0564 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® MluI
λ DN
A, 0
.7%
aga
rose
Note• FastDigest® MboII is blocked by overlapping
dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
• FastDigest® MboII produces DNA fragments that have a single-base 3’-extension which are more difficult to ligate than blunt-ended fragments.
• FastDigest® MboII may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.
Digestion time with 1 µl of FastDigest® MboII bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 65°C, 5 min 16 hours
5’...G A A G A(N)8↓...3’
3’...C T T C T(N)7↑...5’
#FD0824 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA dam– in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam: may overlap – blocked (p.176).Dcm, CpG, EcoKI, EcoBI – no effect.
FastDigest® MboII
λ DN
A (d
am–),
1.4%
aga
rose
130
clea
vage
site
s8
clea
vage
site
s7
clea
vage
site
s
46
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® MreI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
no cleavage sites 5 min 5 min 5 min 3 bp 80°C, 5 min 16 hours
5’...C G↓ C C G G C G...3’
3’...G C G G C C↑G C...5’
#FD2024 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of linearized pJET1 DNA with inserted MreI recognition site in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® MreI
pJET
1-M
reI D
NA/E
am11
05I, 1
.0%
aga
rose
Digestion time with 1 µl of FastDigest® MlyI (SchI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 30 min 5 min 2 bp 80°C, 5 min 1 hour
5’...G A G T C(N)5↓...3’
3’...C T C A G(N)5↑...5’
#FD1374 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® MlyI (SchI)
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® MnlI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 65°C, 5 min 16 hours
5’...C C T C(N)7↓...3’
3’...G G A G(N)6↑...5’
#FD1074 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
FastDigest® MnlI
λ DN
A, 1
.4%
aga
rose
NoteFastDigest® MnlI produces DNA fragments that have a single-base 3’-extension which are more difficult to ligate than blunt-ended fragments.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – blocked (p.181).
61 c
leav
age
site
s26
2 cl
eava
ge s
ites
1 cle
avag
e sit
e
47
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® MslI (RseI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 4 bp 65°C, 20 min 16 hours
5’...C A Y N N↓ N N R T G...3’
3’...G T R N N↑N N Y A C...5’
#FD2004 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG – no effect.EcoKI: may overlap – blocked (p.181).EcoBI: may overlap – effect not determined.
FastDigest® MslI (RseI)
λ DN
A, 1
.2%
aga
rose
Digestion time with 1 µl of FastDigest® MscI (MlsI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 30 min 20 min 3 bp 80°C, 20 min 16 hours
5’...T G G↓C C A...3’
3’...A C C↑G G T...5’
#FD1214 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA dcm– in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, CpG, EcoKI, EcoBI – no effect.Dcm: may overlap – blocked (p.177).
FastDigest® MscI (MlsI)
λ DN
A (d
cm–),
0.7%
aga
rose
NoteFastDigest® MscI (MlsI) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
pBR3
22 D
NA, 1
.0%
aga
rose
Digestion time with 1 µl of FastDigest® MseI (SaqAI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 4 bp 65°C, 5 min 16 hours
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pBR322 DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – effect not determined.
FastDigest® MseI (SaqAI)
5’...T↓T A A...3’
3’...A A T↑T...5’
#FD2174 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
18 c
leav
age
site
s15
cle
avag
e si
tes
62 c
leav
age
site
s
48
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® MssI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 65°C, 10 min 16 hours
5’...G T T T↓A A A C...3’
3’...C A A A↑T T T G...5’
#FD1344 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/HindIII fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – blocked (p.181).
FastDigest® MssI
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® MspI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp No 16 hours
5’...C↓C G G...3’
3’...G G C↑C...5’
#FD0544 400 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
FastDigest® MspI
λ DN
A, 1
.4%
aga
rose
NoteFastDigest® MspI is an isoschizomer of HpaII. When the external C in the sequence CCGG is methylated, these enzymes cannot cleave. However, unlike HpaII, FastDigest® MspI can cleave the sequence when the internal C residue is methylated.
Digestion time with 1 µl of FastDigest® MvaI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 10 min 4 bp No 1 hour
5’...C C↓W G G...3’
3’...G G W↑C C...5’
#FD0554 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
FastDigest® MvaI
λ DN
A, 1
.4%
aga
rose
328
clea
vage
site
s2
clea
vage
site
s70
cle
avag
e si
tes
49
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® Mva1269I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 65°C, 5 min 16 hours
5’...G A A T G C N↓...3’
3’...C T T A C↑G N ...5’
#FD0964 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® Mva1269I
λ DN
A, 1
.0%
aga
rose
Digestion time with 1 µl of FastDigest® NciI (BcnI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 80°C, 20 min 16 hours
5’...C C↓S G G...3’
3’...G G S↑C C...5’
#FD0064 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – cleavage impaired (p.178).
FastDigest® NciI (BcnI)
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® NaeI (PdiI ) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
not determined 5 min 5 min 5 min 3 bp 65°C, 15 min 16 hours
5’...G C C↓G G C...3’
3’...C G G↑C C G...5’
#FD1524 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pBR322 DNA/NdeI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
FastDigest® NaeI (PdiI)
pBR3
22 D
NA/N
deI,
0.7%
aga
rose
Note• For cleavage with FastDigest® NaeI (PdiI) at
least two copies of its recognition sequence are required.
• Certain sites in pBR322 are difficult to cleave with FastDigest® NaeI (PdiI), the same as with its prototype NaeI.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
46 c
leav
age
site
s4
clea
vage
site
s11
4 cl
eava
ge s
ites
50
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® NdeI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 60 min 30 min 3 bp 65°C, 5 min 6 hours
5’...C A↓T A T G...3’
3’...G T A T↑A C...5’
#FD0583 100 µl #FD0584 300 µl
Both supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
#FD0585 1000 µlSupplied with:10X FastDigest® Buffer 2X1 ml10X FastDigest® Green Buffer 2X1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
FastDigest® NdeI
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® NcoI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 10 min 10 min 5 min 3 bp 65°C, 15 min 16 hours
5’...C↓C A T G G...3’
3’...G G T A C↑C...5’
#FD0573 20 µl #FD0574 100 µl#FD0575 300 µl
All supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
FastDigest® NcoI
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest ® NheI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 15 min 5 min 5 min 5 bp 65°C, 5 min 6 hours
5’...G↓C T A G C...3’
3’...C G A T C↑G...5’
#FD0973 50 µl#FD0974 100 µl
Both supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/HindIII fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
FastDigest® NheI
λ DN
A 0.
7% a
garo
se
4 cl
eava
ge s
ites
7 cl
eava
ge s
ites
1 cl
eava
ge s
ite
51
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® NmuCI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 10 min 2 bp 65°C, 5 min 16 hours
5’...↓G T S A C ...3’
3’... C A S T G↑...5’
#FD1514 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – effect not determined.
FastDigest® NmuCI
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® NlaIV (BspLI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 65°C, 20 min 16 hours
5’...G G N↓N C C...3’
3’...C C N↑N G G...5’
#FD1154 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179).
FastDigest® NlaIV (BspLI)
λ DN
A, 1
.4%
aga
rose Note
FastDigest® NlaIV (BspLI) cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Digestion time with 1 µl of FastDigest® NlaIII (Hin1II) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 10 min 5 min 10 min 4 bp 80°C, 5 min 16 hours
5’... C A T G↓ ...3’
3’...↑G T A C ...5’
#FD1834 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® NlaIII (Hin1II)
λ DN
A, 1
.4%
aga
rose
NoteMore stable when stored at -70°C. At -20°C the half-life of FastDigest® NlaIII (Hin1II) is 6 months.
181
clea
vage
site
s82
cle
avag
e si
tes
81 c
leav
age
site
s
52
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® NsiI (Mph1103I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 10 min 3 bp 65°C, 15 min 6 hours
5’...A T G C A↓T...3’
3’...T↑A C G T A...5’
#FD0734 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® NsiI (Mph1103I)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® NruI (RruI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 5 bp No 4 hours
5’...T C G↓C G A...3’
3’...A G C↑G C T...5’
#FD2154 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoBI, EcoKI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® NruI (RruI)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® NotI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
no cleavage sites 30 min 5 min 10 min 2 bp 80°C, 5 min 16 hours
5’...G C↓G G C C G C...3’
3’...C G C C G G↑C G...5’
#FD0593 20 µl #FD0594 50 µl#FD0596 250 µl
All supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pTZ19RJL2 DNA/BseLI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® NotI
pTZ1
9RJL
2/Bs
eLI D
NA, 1
.0%
aga
rose
1 cl
eava
ge s
ite5
clea
vage
site
s14
cle
avag
e si
tes
53
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® PdmI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 65°C, 5 min 16 hours
5’...G A A N N↓N N T T C...3’
3’...C T T N N↑N N A A G...5’
#FD1534 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI: may overlap – effect not determined.
FastDigest® PdmI
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® NspI (XceI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 65°C, 5 min 16 hours
5’...R C A T G↓Y...3’
3’...Y↑G T A C R...5’
#FD1474 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI, CpG – no effect.
FastDigest® NspI (XceI)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® PacI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
no cleavage sites 5 min 5 min 5 min 2 bp 65°C, 10 min 16 hours
5’...T T A A T↓T A A...3’
3’...A A T↑T A A T T...5’
#FD2204 25 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of linear-ized pJET1 DNA with inserted PacI recognition site in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoBI, CpG – no effect.EcoKI: may overlap – effect not determined.
FastDigest® PacI
pJET
1-Pa
cI D
NA/S
daI,
1.0%
aga
rose
32 c
leav
age
site
s1
clea
vage
site
24 c
leav
age
site
s
54
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® PmlI (Eco72I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 80°C, 10 min 1 hour
5’...C A C↓G T G...3’
3’...G T G↑C A C...5’
#FD0364 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not determined.
FastDigest® PmlI (Eco72I)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® PfoI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 65°C, 5 min 16 hours
5’...T↓C C N G G A...3’
3’...A G G N C C↑T...5’
#FD1754 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam: may overlap – cleavage impaired (p.176).Dcm, CpG: may overlap – blocked (p.177, 179).EcoKI, EcoBI – no effect.
FastDigest® PfoI
λ DN
A, 0
.7%
aga
rose
NoteFastDigest® PfoI cleavage is impaired by overlapping dam methylation and blocked by overlapping dcm methylation. To avoid dam or dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Digestion time with 1 µl of FastDigest® PflMI (Van91I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 65°C, 10 min 6 hours
5’...C C A N N N N↓N T G G...3’
3’...G G T N↑N N N N A C C...5’
#FD0714 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, CpG, EcoKI, EcoBI – no effect.Dcm: may overlap – blocked (p.177).
FastDigest® PflMI (Van91I)
λ DN
A, 0
.7%
aga
rose
NoteFastDigest® PflMI (Van91I) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
14 c
leav
age
site
s15
cle
avag
e si
tes
3 cl
eava
ge s
ites
55
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® PsiI (AanI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 5 bp 65°C, 5 min 16 hours
5’...T T A↓T A A ...3’
3’...A A T↑A T T ...5’
#FD2064 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – effect not determined.
FastDigest® PsiI (AanI)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® PshAI (BoxI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 80°C, 20 min 16 hours
5’...G A C N N↓N N G T C...3’
3’...C T G N N↑N N C A G...5’
#FD1434 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – effect not determined.
FastDigest® PshAI (BoxI)
λ DN
A, 1
.0%
aga
rose
Digestion time with 1 µl of FastDigest® PpuMI (Psp5II) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 20 min 2 bp 80°C, 5 min 1 hour
5’...R G↓G W C C Y...3’
3’...Y C C W G↑G R...5’
#FD0764 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/CpoI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, CpG, EcoKI – no effect.Dcm: may overlap – blocked (p.177).EcoBI: may overlap – effect not determined.
FastDigest® PpuMI (Psp5II)
λ DN
A, 0
.7%
aga
rose Note
FastDigest® PpuMI (Psp5II) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).3
clea
vage
site
s7
clea
vage
site
s12
cle
avag
e si
tes
56
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® PsuI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 80°C, 5 min 16 hours
5’...R↓G A T C Y...3’
3’...Y C T A G↑R...5’
#FD1554 150 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® PsuI
λ DN
A, 0
.7%
aga
rose
Note• Surrounding sequences: the presence of
adjacent runs of G-C base pairs confers sig-nificant resistance to cleavage (Armstrong, K. and Bauer, W.R., NAR, 10, 993-1007, 1982).
• FastDigest® PstI will not cut AGCTGCAG when methylated by AluI methyltransferase.
Digestion time with 1 µl of FastDigest® PstI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 30 min 5 min 3 bp No 16 hours
5’...C T G C A↓G...3’
3’...G↑A C G T C...5’
#FD0614 800 µlSupplied with:10X FastDigest® buffer 2x1 ml10X FastDigest® Green Buffer 2x1 ml
#FD0615 2500 µlSupplied with:10X FastDigest® buffer 5x1 ml10X FastDigest® Green Buffer 5x1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
FastDigest® PstI
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® PspFI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 4 80°C, 5 min 16 hours
5’...C C C A G↓C...3’
3’...G↑G G T C G...5’
#FD2224 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
λ DN
A, 0
.7%
aga
rose
FastDigest® PspFI
32 c
leav
age
site
s28
cle
avag
e si
tes
21 c
leav
age
site
s
57
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® PvuII bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp No 0.5 hour
5’...C A G↓C T G...3’
3’...G T C↑G A C...5’
#FD0634 400 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® PvuII
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® PsyI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 80°C, 5 min 4 hours
5’...G A C N↓N N G T C...3’
3’...C T G N N↑N C A G...5’
#FD1334 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/SmaI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® PsyI
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® PvuI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 15 min 5 min 5 min 4 bp 80°C, 5 min 16 hours
5’...C G A T↓C G...3’
3’...G C↑T A G C...5’
#FD0624 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/CpoI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® PvuI
λ DN
A, 0
.7%
aga
rose
2 cl
eava
ge s
ites
3 cl
eava
ge s
ites
15 c
leav
age
site
s
58
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® SacI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 15 min 30 min 10 min 1 bp 65°C, 5 min 16 hours
5’...G A G C T↓C...3’
3’...C↑T C G A G...5’
#FD1133 100 µl#FD1134 200 µl
Both supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/HindIII fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
FastDigest® SacI
λ DN
A, 0
.7%
aga
rose
Note• FastDigest® SacI is sensitive to cytosine
methylation at GAGmCTC but not GAGCTmC and insensitive to adenine methylation at GmAGCTC. AluI methyltransferase (AGmCT) can be used to block FastDigest® SacI.
• FastDigest® SacI is inhibited by common clini-cal anticoagulants found in some preparations of anticoagulated peripheral blood and bone marrow. (Coad, J.E., et al., Inhibition of restric-tion endonucleases by common clinical antico-agulants, Anal. Biochem., 205, 368-369,1992).
Digestion time with 1 µl of FastDigest® RsrII (CpoI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp 65°C, 5 min 16 hours
5’...C G↓G W C C G...3’
3’...G C C W G↑G C...5’
#FD0744 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® RsrII (CpoI)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® RsaI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp No 16 hours
5’...G T↓A C...3’
3’...C A↑T G...5’
#FD1124 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
FastDigest® RsaI
λ DN
A, 1
.4%
aga
rose
113
clea
vage
site
s5
clea
vage
site
s2
clea
vage
site
s
59
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® SapI (LguI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 65°C, 5 min 16 hours
5’...G C T C T T C(N)1↓...3’
3’...C G A G A A G(N)4↑...5’
#FD1934 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® SapI (LguI)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® SalI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 60 min 5 min 3 bp 65°C, 10 min 16 hours
5’...G↓T C G A C...3’
3’...C A G C T↑G...5’
#FD0644 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/Eco81I fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
FastDigest® SalI
λ DN
A, 0
.7%
aga
rose
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion time with 1 µl of FastDigest® SanDI (KflI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 10 min 5 bp No 16 hours
5’...G G↓G W C C C...3’
3’...C C C W G↑G G...5’
#FD2164 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of linear-ized pJET1 DNA with inserted SanDI recognition sites in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm: may overlap – effect not determined.CpG: may overlap – cleavage impaired (p.179).
FastDigest® SanDI (KflI)
pJET
1-Sa
nDI D
NA/B
amHI
, 0.7
% a
garo
se
2 cl
eava
ge s
ites
3 cle
avag
e sit
es10
cle
avag
e si
tes
60
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® SbfI (SdaI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp No 1 hour
5’...C C T G C A↓G G...3’
3’...G G↑A C G T C C...5’
#FD1194 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
FastDigest® SbfI (SdaI)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® Sau96I (Cfr13I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp No 16 hours
5’...G↓G N C C...3’
3’...C C N G↑G...5’
#FD0194 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – blocked (p.177, 179).
FastDigest® Sau96I (Cfr13I)
λ DN
A, 1
.0%
aga
rose
NoteFastDigest® Sau96I (Cfr13I) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Digestion time with 1 µl of FastDigest® Sau3AI (Bsp143I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 10 min 5 min 4 bp 65°C, 20 min 16 hours
5’...↓G A T C ...3’
3’... C T A G↑...5’
#FD0784 40 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – blocked (p.179).
FastDigest® Sau3AI (Bsp143I)
λ DN
A, 1
.4%
aga
rose
NoteFastDigest® DpnI, FastDigest® Sau3AI (Bsp143I) and FastDigest® MboI all recognize the same sequence but have different methylation sensi-tivities (pp.176-181) and cleavage positions.
116
clea
vage
site
s74
cle
avag
e si
tes
5 cl
eava
ge s
ites
61
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® SexAI (CsiI ) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 10 min 5 min 15 min 4 bp 65°C, 5 min 16 hours
5’...A↓C C W G G T...3’
3’...T G G W C C↑A...5’
#FD2114 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG – no effect.EcoBI, EcoKI: may overlap – effect not determined.
FastDigest® SexAI (CsiI)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® ScrFI (Bme1390I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 4 bp No 16 hours
5’...C C↓N G G...3’
3’...G G N↑C C...5’
#FD1424 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA dcm– in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – blocked (p.177, 179).
FastDigest® ScrFI (Bme1390I)
λ DN
A (d
cm–),
1.4%
aga
rose
NoteFastDigest® ScrFI (Bme1390I) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Digestion time with 1 µl of FastDigest® ScaI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 4 bp 65°C, 10 min 16 hours
5’...A G T↓A C T...3’
3’...T C A↑T G A...5’
#FD0434 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – blocked (p.181).
FastDigest® ScaI
λ DN
A, 0
.7%
aga
rose
5 cl
eava
ge s
ites
184
clea
vage
site
s5
clea
vage
site
s
62
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® SfcI (BfmI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp 65°C, 20 min 16 hours
5’...C↓T R Y A G...3’
3’...G A Y R T↑C...5’
#FD1164 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
FastDigest® SfcI (BfmI)
λ DN
A,1.
0% a
garo
se
Note• FastDigest® SfiI cleavage is impaired by
overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
• For cleavage with FastDigest® SfiI at least two copies of its recognition sequence are required. The two sites can be on either the same or different DNA molecules. (Wertzell, L.M. et al., J. Mol. Biol., 248, 581-595, 1995.)
Digestion time with 1 µl of FastDigest® SfiI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
no cleavage sites 15 min 15 min 15 min 5 bp No 16 hours
5’...G G C C N N N N↓N G G C C...3’
3’...C C G G N↑N N N N C C G G...5’
#FD1824 150 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pUC19 DNA with inserted SfiI recognition sites in 15 min at 50°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 50°C.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179). pU
C19-
SfiI D
NA, 0
.7%
aga
rose
FastDigest® SfiI
Digestion time with 1 µl of FastDigest® SfaNI (BmsI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp 65°C, 5 min 16 hours
5’...G C A T C(N)5↓...3’
3’...C G T A G(N)9↑...5’
#FD2124 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – effect not determined.
FastDigest® SfaNI (BmsI)
λ DN
A, 1
.4%
aga
rose Note
At least two copies of FastDigest® SfaNI (BmsI) recognition site are required for efficient cleavage.
169
clea
vage
site
s38
cle
avag
e si
tes
2 cl
eava
ge s
ites
63
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® SpeI (BcuI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
no cleavage sites 5 min 5 min 5 min 1 bp No 16 hours
5’...A↓C T A G T...3’
3’...T G A T C↑A...5’
#FD1253 20 µl #FD1254 50 µl
Both supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pUC19 DNA with inserted BcuI recognition site/Psp1406I fragments in 5 min at 37°C in 1X FastDigest® Buffer. The control DNA is.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG – no effect.EcoKI, EcoBI: may overlap – effect not determined.
FastDigest® SpeI (BcuI)
pUC1
9-Bc
uI DN
A/Ps
p140
6I, 1
.0%
aga
rose
Digestion time with 1 µl of FastDigest® SnaBI (Eco105I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 65°C, 5 min 16 hours
5’...T A C↓G T A...3’
3’...A T G↑C A T...5’
#FD0404 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/CpoI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® SnaBI (Eco105I)
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® SmaI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 1 bp 65°C, 5 min 16 hours
5’...C C C↓G G G...3’
3’...G G G↑C C C...5’
#FD0663 100 µl#FD0664 200 µl
Both supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/Eco81I fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® SmaI
λ DN
A, 0
.7%
aga
rose
3 cl
eava
ge s
ites
1 cl
eava
ge s
ite1
cleav
age
site
64
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® StuI (Eco147I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp 80°C, 10 min 16 hours
5’...A G G↓C C T...3’
3’...T C C↑G G A...5’
#FD0424 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/Eco81I fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, CpG, EcoKI – no effect.Dcm: may overlap – blocked (p.177).EcoBI: may overlap – effect not determined.
FastDigest® StuI (Eco147I)
λ DN
A, 0
.7%
aga
rose Note
FastDigest® StuI (Eco147I) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Digestion time with 1 µl of FastDigest® SspI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 30 min 2 bp 65°C, 5 min 1 hour
5’...A A T↓A T T...3’
3’...T T A↑T A A...5’
#FD0774 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
FastDigest® SspI
λ DN
A, 1
.0%
aga
rose
Digestion time with 1 µl of FastDigest® SphI (PaeI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 5 bp 65°C, 5 min 16 hours
5’...G C A T G↓C...3’
3’...C↑G T A C G...5’
#FD0604 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – blocked (p.181).
FastDigest® SphI (PaeI)
λ DN
A, 0
.7%
aga
rose
6 cl
eava
ge s
ites
20 c
leav
age
site
s6
clea
vage
site
s
65
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® TaaI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp No 16 hours
5’...A C N↓G T...3’
3’...T G↑N C A...5’
#FD1364 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 65°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 65°C.
Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI: may overlap – effect not determined.
FastDigest® TaaI
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® StyI (Eco130I) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 20 min 20 min 3 bp 65°C, 5 min 16 hours
5’...C↓C W W G G...3’
3’...G G W W C↑C...5’
#FD0414 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
FastDigest® StyI (Eco130I)
λ DN
A, 1
.0%
aga
rose
Digestion time with 1 µl of FastDigest® SwaI (SmiI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
no cleavage sites 20 min 30 min 30 min 2 bp 65°C, 15 min 1 hour
5’...A T T T↓A A A T...3’
3’...T A A A↑T T T A...5’
#FD1244 200 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of M13mp18 DNA/MvaI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® SwaI (SmiI)
M13
mp1
8 DN
A/M
vaI,
1.0%
aga
rose
10 c
leav
age
site
s1
clea
vage
site
187
clea
vage
site
s
66
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® TatI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
15 min 15 min 15 min 20 min 5 bp No 1 hour
5’...W↓G T A C W...3’
3’...W C A T G↑W...5’
#FD1294 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 15 min at 65°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 65°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® TatI
λ DN
A, 1
.0%
aga
rose
Digestion time with 1 µl of FastDigest® TaqI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 4 bp No 16 hours
5’...T↓C G A...3’
3’...A G C↑T...5’
#FD0674 400 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA dam– in 5 min at 65°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 65°C.
FastDigest® TaqI
λ DN
A (d
am–),
1.4%
aga
rose
NoteFastDigest® TaqI is blocked by overlapping dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Methylation EffectsDam: may overlap – blocked (p.176).Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion time with 1 µl of FastDigest® TaiI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp No 6 hours
5’... A C G T↓...3’
3’...↑T G C A ...5’
#FD1144 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 65°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 65°C.
Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not determined.
FastDigest® TaiI
λ DN
A, 1
.4%
aga
rose
143
clea
vage
site
s12
1 cl
eava
ge s
ites
24 c
leav
age
site
s
67
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® Tru1I bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 10 min 3 bp No 2 hours
5’...T↓T A A ...3’
3’...A A T↑T...5’
#FD0984 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 65°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 65°C.
Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – blocked (p.181).
FastDigest® Tru1I
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® TfiI (PfeI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 10 min 5 min 2 bp 65°C, 5 min 16 hours
5’...G↓A W T C...3’
3’...C T W A↑G...5’
#FD1784 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – blocked (p.179).EcoBI: may overlap – effect not determined.
FastDigest® TfiI (PfeI)
λ DN
A, 1
.4%
aga
rose
Digestion time with 1 µl of FastDigest® TauI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
15 min 60 min 15 min 30 min 5 bp No 16 hours
5’...G C S G↓C...3’
3’...C↑G S C G...5’
#FD1654 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 15 min at 55°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 55°C.
Methylation EffectsDam, Dcm, EcoBI, EcoKI – no effect.CpG: completely overlaps – blocked (p.178).
FastDigest® TauI
λ DN
A, 1
.4%
aga
rose
NoteFastDigest® TauI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.
181
clea
vage
site
s87
cle
avag
e si
tes
195
clea
vage
site
s
68
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Digestion time with 1 µl of FastDigest® XapI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 10 min 4 bp 80°C, 5 min 6 hours
5’...R↓A A T T Y...3’
3’...Y T T A A↑R...5’
#FD1383 50 µl#FD1384 100 µl
Both supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
FastDigest® XapI
λ DN
A, 0
.7%
aga
rose
Digestion time with 1 µl of FastDigest® TspRI (TscAI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 2 bp No 6 hours
5’... N N C A S T G N N↓...3’
3’...↑N N G T S A C N N ...5’
#FD2104 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 65°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 65°C.
Methylation EffectsDam, Dcm, CpG – no effect.EcoKI, EcoBI: may overlap – effect not determined.
FastDigest® TspRI (TscAI)
λ DN
A, 1
.4%
aga
rose
Note• FastDigest® TspRI (TscAI) produces DNA
fragments with a 9-base 3’-extension. This may result in atypical DNA band patterns.
• FastDigest® TspRI (TscAI) may remain associated with the cleaved DNA. This may cause DNA band shifting during electropho-resis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.
Digestion time with 1 µl of FastDigest® Tsp509I (TasI) bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 5 min 3 bp No 6 hours
5’...↓A A T T ...3’
3’... T T A A↑...5’
#FD1354 100 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pBR322 DNA in 5 min at 65°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 65°C.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – blocked (p.181).
FastDigest® Tsp509I (TasI)
pBR3
22 D
NA, 1
.0%
aga
rose
8 cl
eava
ge s
ites
119
clea
vage
site
s58
cle
avag
e si
tes
69
Protocols and Recommendations p.70
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion time with 1 µl of FastDigest® XbaI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 10 min 2 bp 65°C, 20 min 16 hours
5’...T↓C T A G A...3’
3’...A G A T C↑T...5’
#FD0684 300 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
#FD0685 750 µlSupplied with:10X FastDigest® Buffer 2X1 ml10X FastDigest® Green Buffer 2X1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA dam–/SmaI fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam: may overlap – blocked (p.176).Dcm, CpG, EcoKI, EcoBI – no effect.
FastDigest® XbaI
λ DN
A (d
am–),
0.7%
aga
rose
NoteFastDigest® XbaI is blocked by overlapping dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Digestion time with 1 µl of FastDigest® XhoI bp from end of DNA required for complete digestion
Thermal inactivation
Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl
5 min 5 min 5 min 10 min 2 bp 80°C, 5 min 16 hours
5’...C↓T C G A G...3’
3’...G A G C T↑C...5’
#FD0694 400 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml
#FD0695 1200 µlSupplied with:10X FastDigest® Buffer 3X1 ml10X FastDigest® Green Buffer 3X1 ml
Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/HindIII fragments in 5 min at 37°C in 1X FastDigest® Buffer.
Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – cleavage impaired (p.178).
FastDigest® XhoI
λ DN
A, 0
.7%
aga
rose
1 cl
eava
ge s
ite1
clea
vage
site
70
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Protocols and Recommendations
1.1. Fast DNA digestion1. Prepare the reaction mixture at room tem-
perature in the order indicated:
ComponentVolume
Plasmid DNA
PCR product
Genomic DNA
Water, nuclease-free* (#R0581)
15 µl 17 µl 30 µl
10X FastDigest® Buffer or 10X FastDigest® Green Buffer
2 µl 2 µl ** 5 µl
DNA* 2 µl (up to 1 µg)
10 µl (~0.2 µg)
10 µl (5 µg)
FastDigest® enzyme 1 µl 1 µl 5 µl
Total volume 20 µl 30 µl 50 µl
2. Mix gently and spin down.3. Incubate at 37°C in a heat block or water
thermostat for 5-15 min.***4. Inactivate the enzyme (optional).***Note* The volume of water should be corrected
to keep the indicated total reaction volume. The volume of DNA can be scaled up to 10 µl or down to 0.5 µl depending on the DNA concentration.
** Only 2 µl of 10X FastDigest® buffer or 10X FastDigest® Green Buffer is required for unpurified PCR product in a 30 µl reac-tion volume.
*** See the Certificate of Analysis or Table 1.3 (p.71) for enzyme and substrate specific incubation time, recommended tem-perature and enzyme inactivation conditions.
• For fast simultaneous plasmid vector linearization and dephosphorylation protocol see p.338.
1.2. Reaction set-up for diges-tion of multiple DNA samples1. Pipette 2 µl of each DNA* sample into
labeled tubes.2. Prepare a master mix for n+1 samples.
Example of master mix (for 10 samples of plasmid DNA):
Water, nuclease-free* (#R0581) (10 + 1) x 15 µl = 165 µl
10X FastDigest® Buffer or 10X FastDigest® Green Buffer
(10 + 1) x 2 µl = 22 µl
FastDigest® enzyme (10 + 1) x 1 µl = 11 µl
3. Add 18 µl of master mix* into tubes contain-ing DNA.
Note* The volume of DNA can be scaled up to
10 µl or down to 0.5 µl depending on the DNA concentration. The volume of water and master mix should be corrected to keep the indicated total reaction volume.
1.3. Double and multiple diges-tion of DNA FastDigest® enzymes allow simultaneous digestion of DNA with two or more enzymes in one digestion reaction.• Use 1 µl of each enzyme and scale up the
reaction conditions appropriately.• The combined volume of all added enzymes
should not exceed 1/10 of the total reaction volume.
• If enzymes require different incubation tem-perature perform sequential DNA cleavage: complete the first digestion reaction at the lower temperature, add the second enzyme and increase the digestion temperature for the second enzyme cleavage.
1.4. Scaling up DNA digestion reaction
DNA 1 µg 2 µg 3 µg 4 µg 5 µg
FastDigest® enzyme 1 µl 2 µl 3 µl 4 µl 5 µl
10X FastDigest® Buffer or 10X FastDigest® Green Buffer
2 µl 2 µl 3 µl 4 µl 5 µl
Total volume 20 µl 20 µl 30 µl 40 µl 50 µl
71
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Reaction Conditions
Table 1.3. Reaction conditions for FastDigest® restriction enzymes.
(continued on next page)
FastDigest® restriction enzyme
Specificity 5’→3’
Reaction temp.
Digestion time with 1 µl of FastDigest® enzyme, min bp from end of
DNA required for complete
digestion
Thermalinactivation
Incubationtime
withoutstar
activity
Lambda DNA
1µg/20µl
Plasmid DNA
1µg/20µl
PCR product
~0.2µg/30µl
Genomic DNA
1µg/10µl
FastDigest® AatII GACGT↓C 37°C 15 20 15 15 5 80°C, 5 min 16 h
FastDigest® AccI (XmiI) GT↓MKAC 37°C 5 5 60 5 2 65°C, 5 min 16 h
FastDigest® Acc65I G↓GTACC 37°C 5 5 5 5 3 65°C, 5 min 16 h
FastDigest® AciI (SsiI) CCGC(-3/-1)↓ 37°C 5 5 5 5 2 65°C, 5 min 4 h
FastDigest® AclI (Psp1406I) AA↓CGTT 37°C 5 5 5 5 3 65°C, 5 min 16 h
FastDigest® AcuI (Eco57I)* CTGAAG(16/14)↓ 37°C 15 30 15 15 2 65°C, 5 min 16 h
FastDigest® AfeI (Eco47III) AGC↓GCT 37°C 5 5 5 5 4 65°C, 5 min 16 h
FastDigest® AflII (BspTI) C↓TTAAG 37°C 5 5 5 5 4 NO 16 h
FastDigest® AgeI (BshTI) A↓CCGGT 37°C 5 5 5 5 3 80°C, 5 min 16 h
FastDigest® AjuI* ↓(7/12)GAA(N)7TTGG(11/6)↓ 37°C 5 5 10 5 ND 65°C, 5 min 16 h
FastDigest® AleI (OliI) CACNN↓NNGTG 37°C 5 5 10 5 3 65°C, 5 min 6 h
FastDigest® AluI AG↓CT 37°C 15 15 15 15 4 65°C, 5 min 16 h
FastDigest® Alw21I GWGCW↓C 37°C 5 5 5 5 1 80°C, 20 min 16 h
FastDigest® Alw26I GTCTC(1/5)↓ 37°C 5 5 5 5 1 65°C, 5 min 16 h
FastDigest® AlwNI (CaiI) CAGNNN↓CTG 37°C 5 5 5 5 4 65°C, 10 min 16 h
FastDigest® ApaI GGGCC↓C 37°C 5 5 20 10 2 65°C, 5 min 16 h
FastDigest® ApaLI (Alw44I) G↓TGCAC 37°C 5 60 10 5 4 80°C, 5 min 16h
FastDigest® AscI (SgsI) GG↓CGCGCC 37°C 5 5 5 5 2 65°C, 20 min 16 h
FastDigest® AseI (VspI) AT↓TAAT 37°C 5 5 5 30 2 65°C, 5 min 16 h
FastDigest® AsiSI (SfaAI) GCGAT↓CGC 37°C no sites 5 60 5 5 80°C, 5 min 6 h
FastDigest® AvaI (Eco88I) C↓YCGRG 37°C 5 5 5 5 2 65°C, 5 min 16 h
FastDigest® AvaII (Eco47I) G↓GWCC 37°C 5 5 5 5 1 80°C, 20 min 16 h
FastDigest® AvrII (XmaJI) C↓CTAGG 37°C 5 10 5 10 2 NO 16 h
FastDigest® BamHI G↓GATCC 37°C 5 5 5 5 2 80°C, 5 min 1 h
FastDigest® BanI (BshNI) G↓GYRCC 37°C 5 60 5 15 2 65°C, 10 min 16 h
FastDigest® BbsI (BpiI) GAAGAC(2/6)↓ 37°C 5 5 5 5 1 65°C, 10 min 16 h
FastDigest® BbvI (Lsp1109I) GCAGC(8/12)↓ 37°C 5 5 5 10 2 65°C, 5 min 1 h
FastDigest® BclI T↓GATCA 37°C 5 5 15 5 3 80°C, 20 min 16 h
FastDigest® BfaI (FspBI) C↓TAG 37°C 5 15 5 20 3 80°C, 5 min 16 h
FastDigest® BglI GCCNNNN↓NGGC 37°C 5 5 5 5 3 65°C, 5 min 2 h
FastDigest® BglII A↓GATCT 37°C 5 20 30 20 3 NO 16 h
FastDigest® BlpI (Bpu1102I) GC↓TNAGC 37°C 5 5 5 5 2 80°C, 5 min 16 h
FastDigest® Bme1580I (BseSI) GKGCM↓C 37°C 5 5 5 30 3 80°C, 15 min 16 h
FastDigest® BmtI (BspOI) GCTAG↓C 37°C 5 5 15 5 3 80°C, 5min 1 h
FastDigest® BplI* ↓(8/13)GAG(N)5CTC(13/8)↓ 37°C 15 20 30 30 ND 65°C, 5 min 16 h
FastDigest® BpmI (GsuI) CTGGAG(16/14)↓ 30°C 15 30 15 15 2 65°C, 5 min 16 h
FastDigest® Bpu10I CCTNAGC(-5/-2)↓ 37°C 15 15 30 15 3 80°C, 5 min 1 h
FastDigest® BsaAI (Ppu21I) YAC↓GTR 37°C 5 5 5 5 1 65°C, 5 min 6 h
FastDigest® BsaBI (BseJI) GATNN↓NNATC 65°C 5 5 5 5 3 NO 1 h
FastDigest® BsaHI (Hin1I) GR↓CGYC 37°C 5 5 5 5 1 65°C, 5 min 16 h
FastDigest® BsaJI (BseDI) C↓CNNGG 37°C 5 5 5 5 3 80°C, 5 min 16 h
FastDigest® BseGI GGATG(2/0)↓ 37°C 5 5 5 5 2 80°C, 5 min 16 h
FastDigest® BseNI ACTGG(1/-1)↓ 65°C 5 5 5 5 2 80°C, 5 min 16 h
FastDigest® BseXI GCAGC(8/12)↓ 65°C 15 15 15 15 3 80°C, 15 min 16 h
FastDigest® Bsh1236I CG↓CG 37°C 5 5 5 5 1 80°C, 10 min 16 h
FastDigest® BsiEI (Bsh1285I) CGRY↓CG 37°C 15 15 15 15 3 80°C, 15 min 1 h
FastDigest® BsiWI (Pfl23II) C↓GTACG 37°C 15 15 15 15 3 65°C, 5 min 16 h
FastDigest® BslI (BseLI) CCNNNNN↓NNGG 37°C 5 5 5 5 2 NO 16 h
FastDigest® BsmBI (Esp3I)* CGTCTC(1/5)↓ 37°C 5 5 5 5 1 65°C, 10 min 6 h
FastDigest® BsmFI (FaqI)* GGGAC(10/14)↓ 37°C 15 15 15 15 3 65°C, 5 min 16 h
72
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
(continued on next page)
Table 1.3. Reaction conditions for FastDigest® restriction enzymes.
FastDigest® restriction enzyme
Specificity 5’→3’
Reaction temp.
Digestion time with 1 µl of FastDigest® enzyme, min bp from end of
DNA required for complete
digestion
Thermalinactivation
Incubationtime
withoutstar
activity
Lambda DNA
1µg/20µl
Plasmid DNA
1µg/20µl
PCR product
~0.2µg/30µl
Genomic DNA
1µg/10µlFastDigest® Bsp119I TT↓CGAA 37°C 5 5 5 5 1 80°C, 5 min 16 h
FastDigest® Bsp120I G↓GGCCC 37°C 5 5 15 5 3 80°C, 10 min 16 h
FastDigest® Bsp1286I (SduI) GDGCH↓C 37°C 5 5 5 5 2 80°C, 15 min 1 h
FastDigest® Bsp1407I T↓GTACA 37°C 5 5 5 5 3 80°C, 10 min 16 h
FastDigest® BspCNI (BseMII)* CTCAG(10/8)↓ 55°C 15 30 15 15 2 80°C, 5 min 16 h
FastDigest® BspHI (PagI) T↓CATGA 37°C 5 10 5 10 3 80°C, 5 min 0.5 h
FastDigest® BspMI (BveI)* ACCTGC(4/8)↓ 37°C 15 15 60 20 5 65°C, 5 min 16 h
FastDigest® BsrBI (MbiI) CCGCTC(-3/-3)↓ 37°C 5 5 5 5 3 65°C, 5 min 16 h
FastDigest® BsrDI (BseMI) GCAATG(2/0)↓ 55°C 15 15 15 15 3 80°C, 5 min 16 h
FastDigest® BsrFI (Cfr10I) R↓CCGGY 37°C 5 5 10 5 2 NO 2 h
FastDigest® BssHII (PteI) G↓CGCGC 37°C 5 5 5 5 3 80°C, 5 min 16 h
FastDigest® BstXI CCANNNNN↓NTGG 37°C 5 5 5 5 4 80°C, 5 min 0.5 h
FastDigest® BstZ17I (Bst1107I) GTA↓TAC 37°C 5 5 5 10 3 NO 6 h
FastDigest® Bsu36I (Eco81I) CC↓TNAGG 37°C 5 5 5 5 2 80°C, 10 min 16 h
FastDigest® ClaI (Bsu15I) AT↓CGAT 37°C 5 5 5 5 2 65°C, 15 min 16 h
FastDigest® Csp6I G↓TAC 37°C 5 5 5 5 2 80°C, 10 min 16 h
FastDigest® DdeI (HpyF3I) C↓TNAG 37°C 5 5 5 10 3 65°C, 5 min 16 h
FastDigest® DpnI Gm6A↓TC 37°C ND 5not
applicable10 1 80°C, 5 min 16 h
FastDigest® DraI TTT↓AAA 37°C 5 5 5 5 4 65°C, 5 min 16 h
FastDigest® DraIII (AdeI) CACNNN↓GTG 37°C 5 5 5 5 2 80°C, 5 min 6 h
FastDigest® DrdI (AasI) GACNNNN↓NNGTC 37°C 5 5 5 5 1 80°C, 10 min 1 h
FastDigest® EagI (Eco52I) C↓GGCCG 37°C 5 20 20 5 3 65°C, 5 min 16 h
FastDigest® Eam1105I GACNNN↓NNGTC 37°C 5 5 5 5 3 65°C, 5 min 16 h
FastDigest® EarI (Eam1104I) CTCTTC(1/4)↓ 37°C 5 5 5 5 3 80°C, 5 min 6 h
FastDigest® Ecl136II GAG↓CTC 37°C 5 5 5 5 1 65°C, 5 min 6 h
FastDigest® Eco31I GGTCTC(1/5)↓ 37°C 5 5 5 5 3 65°C, 5 min 16 h
FastDigest® Eco91I G↓GTNACC 37°C 5 5 5 5 2 65°C, 10 min 2 h
FastDigest® EcoNI (XagI) CCTNN↓NNNAGG 37°C 5 5 5 20 2 65°C, 5 min 16 h
FastDigest® EcoO109I RG↓GNCCY 37°C 5 10 15 5 2 65°C, 5 min 16 h
FastDigest® EcoRI G↓AATTC 37°C 5 5 20 5 2 80°C, 5 min 0.5 h
FastDigest® EcoRV (Eco32I) GAT↓ATC 37°C 5 5 5 5 2 NO 16 h
FastDigest® EheI GGC↓GCC 37°C 5 5 5 5 2 65°C, 5 min 6 h
FastDigest® Fnu4HI (SatI) GC↓NGC 37°C 5 5 10 5 3 65°C, 5 min 16 h
FastDigest® FokI GGATG(9/13)↓ 37°C 5 5 5 5 1 65°C, 5 min 1 h
FastDigest® FspI (NsbI) TGC↓GCA 37°C 5 5 5 5 3 65°C, 15 min 16 h
FastDigest® FspAI RTGC↓GCAY 37°C 5 5 5 5 4 65°C, 5 min 16 h
FastDigest® HaeII (BfoI) RGCGC↓Y 37°C 5 5 5 5 2 65°C, 10 min 1h
FastDigest® HaeIII (BsuRI) GG↓CC 37°C 5 5 5 5 3 NO 16 h
FastDigest® HgaI (CseI) GACGC(5/10)↓ 37°C 15 15 30 15 2 80°C, 5 min 16 h
FastDigest® HhaI GCG↓C 37°C 5 5 5 5 1 NO 16 h
FastDigest® HincII GTY↓RAC 37°C 5 5 5 10 1 65°C, 5 min 16 h
FastDigest® HindIII A↓AGCTT 37°C 5 5 20 10 3 80°C, 10 min 16 h
FastDigest® HinfI G↓ANTC 37°C 5 5 5 5 2 65°C, 20 min 16 h
FastDigest® HinP1I (Hin6I) G↓CGC 37°C 5 10 5 5 2 80°C, 10 min 16 h
FastDigest® HpaI (KspAI) GTT↓AAC 37°C 5 5 5 5 3 65°C, 20 min 0.5 h
FastDigest® HpaII C↓CGG 37°C 5 5 5 5 3 65°C, 5 min 16 h
FastDigest® Hpy8I GTN↓NAC 37°C 5 5 5 10 2 80°C, 5 min 16 h
FastDigest® HpyF10VI GCNNNNN↓NNGC 37°C 5 5 5 5 2 80°C, 5 min 16 h
FastDigest® KpnI GGTAC↓C 37°C 5 5 5 5 3 80°C, 5 min 16 h
FastDigest® Kpn2I T↓CCGGA 37°C 5 5 5 5 2 80°C, 5 min 16 h
FastDigest® MauBI CG↓CGCGCG 37°C no sites 5 10 5 5 65°C, 5 min 16 h
73
1
1. RESTRICTION ENZYMES®
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.3. Reaction conditions for FastDigest® restriction enzymes.
(continued on next page)
FastDigest® restriction enzyme
Specificity 5’→3’
Reaction temp.
Digestion time with 1 µl of FastDigest® enzyme, min bp from end of
DNA required for complete
digestion
Thermalinactivation
Incubationtime
withoutstar
activity
Lambda DNA
1µg/20µl
Plasmid DNA
1µg/20µl
PCR product
~0.2µg/30µl
Genomic DNA
1µg/10µlFastDigest® MboI ↓GATC 37°C 5 5 5 10 1 65°C, 15 min 16 h
FastDigest® MboII GAAGA(8/7)↓ 37°C 5 5 5 5 2 65°C, 5 min 16 h
FastDigest® MfeI (MunI) C↓AATTG 37°C 5 5 5 5 3 NO 16 h
FastDigest® MluI A↓CGCGT 37°C 5 5 5 15 3 80°C, 5 min 16 h
FastDigest® MlyI (SchI) GAGTC(5/5)↓ 37°C 5 5 30 5 2 80°C, 5 min 1 h
FastDigest® MnlI CCTC(7/6)↓ 37°C 5 5 5 5 2 65°C, 5 min 16 h
FastDigest® MreI CG↓CCGGCG 37°C no sites 5 5 5 3 80°C, 5 min 16 h
FastDigest® MscI (MlsI) TGG↓CCA 37°C 5 5 30 20 3 80°C, 20 min 16 h
FastDigest® MseI (SaqAI) T↓TAA 37°C 5 5 5 5 4 65°C, 5 min 16 h
FastDigest® MslI (RseI) CAYNN↓NNRTG 37°C 5 5 5 5 4 65°C, 20 min 16 h
FastDigest® MspI C↓CGG 37°C 5 5 5 5 3 NO 16 h
FastDigest® MssI GTTT↓AAAC 37°C 5 5 5 5 2 65°C, 10 min 16 h
FastDigest® MvaI CC↓WGG 37°C 5 5 5 10 4 NO 1 h
FastDigest® Mva1269I GAATGC(1/-1)↓ 37°C 5 5 5 5 3 65°C, 5 min 16 h
FastDigest® NaeI (PdiI) GCC↓GGC 37°C ND 5 5 5 3 65°C, 15 min 16 h
FastDigest® NciI (BcnI) CC↓SGG 37°C 5 5 5 5 3 80°C, 20 min 16 h
FastDigest® NcoI C↓CATGG 37°C 5 10 10 5 3 65°C, 15 min 16 h
FastDigest® NdeI CA↓TATG 37°C 5 5 60 30 3 65°C, 5 min 6 h
FastDigest® NheI G↓CTAGC 37°C 5 15 5 5 5 65°C, 5 min 6 h
FastDigest® NlaIII (Hin1II) CATG↓ 37°C 5 10 5 10 4 80°C, 5 min 16 h
FastDigest® NlaIV (BspLI) GGN↓NCC 37°C 5 5 5 5 2 65°C, 20 min 16 h
FastDigest® NmuCI ↓GTSAC 37°C 5 5 5 10 2 65°C, 5 min 16 h
FastDigest® NotI GC↓GGCCGC 37°C no sites 30 5 10 2 80°C, 5 min 16 h
FastDigest® NruI (RruI) TCG↓CGA 37°C 5 5 5 5 5 NO 4 h
FastDigest® NsiI (Mph1103I) ATGCA↓T 37°C 5 5 5 10 3 65°C, 15 min 6 h
FastDigest® NspI (XceI) RCATG↓Y 37°C 5 5 5 5 2 65°C, 5 min 16 h
FastDigest® PacI TTAAT↓TAA 37°C no sites 5 5 5 2 65°C, 10 min 16 h
FastDigest® PdmI GAANN↓NNTTC 37°C 5 5 5 5 2 65°C, 5 min 16 h
FastDigest® PflMI (Van91I) CCANNNN↓NTGG 37°C 5 5 5 5 3 65°C, 10 min 6 h
FastDigest® PfoI T↓CCNGGA 37°C 5 5 5 5 3 65°C, 5 min 16 h
FastDigest® PmlI (Eco72I) CAC↓GTG 37°C 5 5 5 5 2 80°C, 10 min 1 h
FastDigest® PpuMI (Psp5II) RG↓GWCCY 37°C 5 5 5 20 2 80°C, 5 min 1 h
FastDigest® PshAI (BoxI) GACNN↓NNGTC 37°C 5 5 5 5 3 80°C, 20 min 16 h
FastDigest® PsiI (AanI) TTA↓TAA 37°C 5 5 5 5 5 65°C, 5 min 16 h
FastDigest® PspFI CCCAGC(-1/-5)↓ 37°C 5 5 5 5 4 80°C, 5 min 16 h
FastDigest® PstI CTGCA↓G 37°C 5 5 30 5 3 NO 16 h
FastDigest® PsuI R↓GATCY 37°C 5 5 5 5 2 80°C, 5 min 16 h
FastDigest® PsyI GACN↓NNGTC 37°C 5 5 5 5 2 80°C, 5 min 4 h
FastDigest® PvuI CGAT↓CG 37°C 5 15 5 5 4 80°C, 5 min 16 h
FastDigest® PvuII CAG↓CTG 37°C 5 5 5 5 1 NO 0.5 h
FastDigest® RsaI GT↓AC 37°C 5 5 5 5 3 NO 16 h
FastDigest® RsrII (CpoI) CG↓GWCCG 37°C 5 5 5 5 1 65°C, 5 min 16 h
FastDigest® SacI GAGCT↓C 37°C 5 15 30 10 1 65°C, 5 min 16 h
FastDigest® SalI G↓TCGAC 37°C 5 5 60 5 3 65°C, 10 min 16 h
FastDigest® SanDI (KflI) GG↓GWCCC 37°C 5 5 5 10 5 NO 16 h
FastDigest® SapI (LguI) GCTCTTC(1/4)↓ 37°C 5 5 5 5 2 65°C, 5 min 16 h
FastDigest® Sau3AI (Bsp143I) ↓GATC 37°C 5 5 10 5 4 65°C, 20 min 16 h
FastDigest® Sau96I (Cfr13I) G↓GNCC 37°C 5 5 5 5 2 NO 16 h
FastDigest® SbfI (SdaI) CCTGCA↓GG 37°C 5 5 5 5 3 NO 1 h
FastDigest® ScaI AGT↓ACT 37°C 5 5 5 5 4 65°C, 10 min 16 h
FastDigest® ScrFI (Bme1390I) CC↓NGG 37°C 5 5 5 5 4 NO 16 h
FastDigest® SexAI (CsiI) A↓CCWGGT 37°C 5 10 5 15 4 65°C, 5 min 16 h
74
1. F
astD
iges
t® R
ESTR
ICTI
ON E
NZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.3. Reaction conditions for FastDigest® restriction enzymes.
Note* – for digestion with FastDigest® BsmBI (Esp3I) add 1mM DTT. for digestion with FastDigest® AcuI (Eco57I), FastDigest® AjuI and FastDigest® BspCNI (BseMII) add 0.01 mM SAM. for digestion with FastDigest® BplI and FastDigest® BsmFI (FaqI) add 0.05 mM SAM. for digestion with FastDigest® BspMI (BveI) add 0.5 µM oligonucleotide.NO – no thermal inactivation. Spin column purification or phenol/chloroform extraction and ethanol precipitation of DNA is recommended.
ND – not determined.
Single letter codeR = G or A; H = A, C or T; Y = C or T; V = A, C or G;W = A or T; B = C, G or T;M = A or C; D = A, G or T;K = G or T; N = G, A, T or C.S = C or G;
FastDigest® restriction enzyme
Specificity 5’→3’
Reaction temp.
Digestion time with 1 µl of FastDigest® enzyme, min bp from end of
DNA required for complete
digestion
Thermalinactivation
Incubationtime
withoutstar
activity
Lambda DNA
1µg/20µl
Plasmid DNA
1µg/20µl
PCR product
~0.2µg/30µl
Genomic DNA
1µg/10µlFastDigest® SfaNI (BmsI) GCATC(5/9)↓ 37°C 5 5 5 5 1 65°C, 5 min 16 h
FastDigest® SfcI (BfmI) C↓TRYAG 37°C 5 5 5 5 2 65°C, 20 min 16 h
FastDigest® SfiI GGCCNNNN↓NGGCC 50°C no sites 15 15 15 5 NO 16 h
FastDigest® SmaI CCC↓GGG 37°C 5 5 5 5 1 65°C, 5 min 16 h
FastDigest® SnaBI (Eco105I) TAC↓GTA 37°C 5 5 5 5 3 65°C, 5 min 16 h
FastDigest® SpeI (BcuI) A↓CTAGT 37°C no sites 5 5 5 1 NO 16 h
FastDigest® SphI (PaeI) GCATG↓C 37°C 5 5 5 5 5 65°C, 5 min 16 h
FastDigest® SspI AAT↓ATT 37°C 5 5 5 30 2 65°C, 5 min 1 h
FastDigest® StuI (Eco147I) AGG↓CCT 37°C 5 5 5 5 3 80°C, 10 min 16 h
FastDigest® StyI (Eco130I) C↓CWWGG 37°C 5 5 20 20 3 65°C, 5 min 16 h
FastDigest® SwaI (SmiI) ATTT↓AAAT 37°C no sites 20 30 30 2 65°C, 15 min 1 h
FastDigest® TaaI ACN↓GT 65°C 5 5 5 5 3 NO 16 h
FastDigest® TaiI ACGT↓ 65°C 5 5 5 5 2 NO 6 h
FastDigest® TaqI T↓CGA 65°C 5 5 5 5 4 NO 16 h
FastDigest® TatI W↓GTACW 65°C 15 15 15 20 5 NO 1 h
FastDigest® TauI GCSG↓C 55°C 15 60 15 30 5 NO 16 h
FastDigest® TfiI (PfeI) G↓AWTC 37°C 5 5 10 5 2 65°C, 5 min 16 h
FastDigest® Tru1I T↓TAA 65°C 5 5 5 10 3 NO 2 h
FastDigest® Tsp509I (TasI) ↓AATT 65°C 5 5 5 5 3 NO 6 h
FastDigest® TspRI (TscAI) CASTG(2/-7)↓ 65°C 5 5 5 5 2 NO 6 h
FastDigest® XapI R↓AATTY 37°C 5 5 5 10 4 80°C, 5 min 6 h
FastDigest® XbaI T↓CTAGA 37°C 5 5 5 10 2 65°C, 20 min 16 h
FastDigest® XhoI C↓TCGAG 37°C 5 5 5 10 2 80°C, 5 min 16 h
75
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Activity and Quality Control Assays
Activity AssayOne unit is the amount of enzyme required to hydrolyze 1 µg of substrate DNA in 60 min in 50 µl of reaction mixture under the recom-mended conditions. To determine restriction enzyme activity, concentrated enzymes are first diluted to approximately 0,5-1 units/µl with an enzyme dilution buffer (20 mM potassium phosphate (pH 7.4), 200 mM KCl, 1 mM EDTA, 7 mM 2-mercaptoethanol, 10% glycerol and 0.2 mg/ml BSA) and then used to cleave DNA.In general, enzymes are assayed with λ phage DNA at 37°C. However, some exceptions apply:• Restriction enzymes that show optimum
activity at temperatures other than 37°C are assayed under their optimal temperature.
• Restriction enzymes without recognition sites on λ DNA are assayed with another specific DNA substrate.
• Restriction enzymes with only a few recognition sites on the λ DNA are assayed using λ DNA hydrolyzed with another restriction enzyme.
• Restriction enzymes sensitive to Dam or Dcm methylation are assayed with λ DNA (dam–, dcm–).
Quality Control
Labeled Oligonucleotide (LO) TestThe labelled oligonucleotides are incubated with an excess of restriction enzyme, separated on a polyacrylamide gel under denaturing conditions and analyzed by phospho-imaging. The restric-tion enzyme passes this quality control test if there is no degradation of labelled oligonucle-otides and no decrease in specific radioactivity (see Fig. 1.1 on p.2).
Non-specific Nuclease and Cross-contamination AssayVarying amounts of restriction enzyme (2-20 units) are incubated for 16 hours with 1 µg of substrate DNA under the recommended assay conditions. After electrophoretic separation of the DNA fragments, the characteristic banding patterns are examined for alterations. To pass the test, the restriction enzyme must yield an unaltered banding pattern under conditions of up to 160-fold overdigestion (10 units x 16 hours). For information regarding restriction enzyme star activity, see the product description or Certificate of Analysis supplied with each enzyme.
Ligation and Recleavage AssayThe ligation and re-cleavage assay tests the integrity of DNA ends. DNA fragments obtained after 2-, 10- and 50-fold overdigestion (units/µg of DNA x hours) are ligated with T4 DNA ligase and then recut with the same restriction enzyme. Only DNA fragments with intact 5’- and 3’-termini are ligated by T4 DNA ligase, and only molecules with reconstructed recognition sites can then be cleaved by the same restric-tion enzyme. A restriction enzyme conforms to the quality criterion if the ligation efficiency of DNA fragments generated by digestion with the restriction enzyme doesn’t depend on the fold of DNA overdigestion with the assayed enzyme.The percentage of DNA that can be success-fully ligated and then re-cleaved is presented for each restriction enzyme both in its catalog description and the Certificate of Analysis sup-plied with each enzyme.
Fermentas conventional restriction endonucleases are classic restriction enzymes, which normally digest DNA in one hour and each require a specific reaction buffer for 100% activity.
Storage and ShippingAll conventional restriction enzymes should be stored at -20°C. During shipment on dry ice, enzymes may freeze. This does not affect their quality – all Fermentas enzymes are 100% active after at least three freeze-thaw cycles. For 24-48 hour delivery, enzymes may be shipped on blue ice since their quality is not affected by short exposure to 4°C.
Conventional Restriction Enzymes
Blue/White (B/W) Cloning AssayThe Blue/White cloning assay is designed to test the integrity of DNA ends. pUC57 DNA is digested at unique sites within the lacZ reporter gene with 10 units of a restriction enzyme in its optimal buffer. After a 16 hour incubation, the plasmid DNA is recircularized by ligation and transformed into E.coli XL1-Blue competent cells. The cells are then plated onto X-Gal/IPTG/Amp agar. An intact lacZ gene will produce a blue colony. If the termini of the linearized pUC57 are altered by contaminating exodeoxy-nucleases, the lacZ reading frame is inter-rupted, which results in the appearance of white colonies. The higher the ratio of blue:white colonies, the higher the quality of the restriction enzyme. An enzyme conforms to this quality criterion if the number of white colonies does not exceed 3%. Details of the assay are given in the Certificate of Analysis of each product. The test is applicable for enzymes recognizing unique sites within the lacZ reporter gene, and for those lacking recognition sites in pUC57. In the latter case, the assay is performed with the mixture of pUC57/HindIII, pUC57/PstI and pUC57/Eco32I DNA fragments representing three different types of termini (3’-overhang, 5’-overhang and blunt ends).
76
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Product List and Cross-reference to FastDigest® Restriction Enzymes
Table 1.4. Fermentas conventional restriction enzymes.
Conventional restriction enzyme
FastDigest® restriction enzyme
Prototype Specificity 5’→3’ Cat. # Page
AanI (PsiI) FastDigest® PsiI (AanI) PsiI TTA↓TAA ER2061 80
AarI AarI CACCTGC(4/8)↓ ER1581/2 80AasI (DrdI) FastDigest® DrdI (AasI) DrdI GACNNNN↓NNGTC ER1721 80
AatII FastDigest® AatII AatII GACGT↓C ER0991/2 81
Acc65I (Asp718I) FastDigest® Acc65I KpnI (GGTAC↓C) G↓GTACC ER0901/2 81
AdeI (DraIII ) FastDigest® DraIII (AdeI) DraIII CACNNN↓GTG ER1231 82
AjiI (BmgBI) BtrI CAC↓GTC ER1941 82AjuI FastDigest® AjuI AjuI ↓(7/12)GAA(N)7TTGG(11/6)↓ ER1951 83
AlfI AlfI ↓(10/12)GCA(N)6TGC(12/10)↓ ER1801 83AloI AloI ↓(7/12-13)GAAC(N)6TCC(12-13/7)↓ ER1491 84AluI FastDigest® AluI AluI AG↓CT ER0011/2 84
Alw21I (BsiHKAI) FastDigest® Alw21I HgiAI GWGCW↓C ER0021 84
Alw26I (BsmAI) FastDigest® Alw26I BsmAI GTCTC(1/5)↓ ER0031 85
Alw44I (ApaLI) FastDigest® ApaLI (Alw44I) ApaLI G↓TGCAC ER0041 85
ApaI FastDigest® ApaI ApaI GGGCC↓C ER1411/5 85
BamHI FastDigest® BamHI BamHI G↓GATCC ER0051/2/3/5 86
BauI (BssSI) BsiI CACGAG(-5/-1)↓ ER1841 87BclI FastDigest® BclI BclI T↓GATCA ER0721/2/5 87
BcnI (NciI) FastDigest® NciI (BcnI) CauII CC↓SGG ER0061 88
BcuI (SpeI) FastDigest® SpeI (BcuI) SpeI A↓CTAGT ER1251/2 88
BdaI BdaI ↓(10/12)TGA(N)6TCA(12/10)↓ ER1961 88BfiI (BmrI) BfiI ACTGGG(5/4)↓ ER1591 89
BfmI (SfcI) FastDigest® SfcI (BfmI) SfeI C↓TRYAG ER1161/2 89
BfuI (BciVI) BciVI GTATCC(6/5)↓ ER1501 90BglI FastDigest® BglI BglI GCCNNNN↓NGGC ER0071/2 90
BglII FastDigest® BglII BglII A↓GATCT ER0081/2 90
Bme1390I (ScrFI) FastDigest® ScrFI (Bme1390I) ScrFI CC↓NGG ER1421/2 91
BoxI (PshAI) FastDigest® PshAI (BoxI) PshAI GACNN↓NNGTC ER1431 91
BpiI (BbsI) FastDigest® BbsI (BpiI) BbvII GAAGAC(2/6)↓ ER1011/2 92
BplI FastDigest® BpII BplI ↓(8/13)GAG(N)5CTC(13/8)↓ ER1311/2 92
Bpu10I FastDigest® Bpu10l Bpu10I CCTNAGC(-5/-2)↓ ER1181 93
Bpu1102I (BlpI) FastDigest® BlpI (Bpu1102I) EspI GC↓TNAGC ER0091/2 93
BseDI (BsaJI) FastDigest® BsaJI (BseDI) SecI C↓CNNGG ER1081/2 94
BseGI (BtsCI) FastDigest® BseGI FokI (GGATG(9/13)↓) GGATG(2/0)↓ ER0871/2 94
BseJI (BsaBI) FastDigest® BsaBI (BseJI) BsaBI GATNN↓NNATC ER1711 94
BseLI (BslI) FastDigest® BslI (BseLI) BsiYI CCNNNNN↓NNGG ER1201 95
BseMI (BsrDI) FastDigest® BsrDI (BseMI) BsrDI GCAATG(2/0)↓ ER1261/2 95
BseMII (BspCNI) FastDigest® BspCNI (BseMII) BseMII CTCAG(10/8)↓ ER1401 95
BseNI (BsrI) FastDigest® BseNI BsrI ACTGG(1/-1)↓ ER0881/2 96
BseSI (Bme1580I) FastDigest® Bme1580I (BseSI) BseSI GKGCM↓C ER1441 96
BseXI (BbvI) FastDigest® BseXI BbvI GCAGC(8/12)↓ ER1451/2 96
Bsh1236I (BstUI) FastDigest® Bsh1236I FnuDII CG↓CG ER0921/2 97
Bsh1285I (BsiEI) FastDigest® BsiEI (Bsh1285I) McrI CGRY↓CG ER0891 97
BshNI (BanI) FastDigest® BanI (BshNI) HgiCI G↓GYRCC ER1001 97
BshTI (AgeI) FastDigest® AgeI (BshTI) AgeI A↓CCGGT ER1461/2 98
Bsp68I (NruI) FastDigest® NruI (RruI) NruI TCG↓CGA ER0111 98
Bsp119I (BstBI) FastDigest® Bsp119I AsuII TT↓CGAA ER0121 99
Bsp120I (PspOMI) FastDigest® Bsp120I ApaI (GGGCC↓C) G↓GGCCC ER0131 99
Bsp143I (Sau3AI) FastDigest® Sau3AI (Bsp143I) MboI ↓GATC ER0781/2 99
Bsp1407I (BsrGI) FastDigest® Bsp1407I Bsp1407I T↓GTACA ER0931/2 100
(continued on next page)
77
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.4. Fermentas conventional restriction enzymes.
Conventional restriction enzyme
FastDigest® restriction enzyme
Prototype Specificity 5’→3’ Cat. # Page
BspLI (NlaIV) FastDigest® NlaIV (BspLI) NlaIV GGN↓NCC ER1151/2 100
BspOI (BmtI) FastDigest® BmtI (BspOI) NheI (G↓CTAGC) GCTAG↓C ER2041 100
BspPI (AlwI) BinI GGATC(4/5)↓ ER1321/2 101BspTI (AflII) FastDigest® AflII (BspTI) AflII C↓TTAAG ER0831 101
Bst1107I (BstZ17I) FastDigest® BstZ17I (Bst1107I) SnaI GTA↓TAC ER0701 102
BstXI FastDigest® BstXI BstXI CCANNNNN↓NTGG ER1021/2 102
Bsu15I (ClaI) FastDigest® ClaI (Bsu15I) ClaI AT↓CGAT ER0141/2/5 103
BsuRI (HaeIII) FastDigest® HaeIII (BsuRI) HaeIII GG↓CC ER0151 103
BveI (BspMI) FastDigest® BspMI (BveI) BspMI ACCTGC(4/8)↓ ER1741 104
CaiI (AlwNI) FastDigest® AlwNI (CaiI) AlwNI CAGNNN↓CTG ER1391 104
CfrI (EaeI) CfrI Y↓GGCCR ER0161 105Cfr9I (XmaI) SmaI (CCC↓GGG) C↓CCGGG ER0171/2 105Cfr10I (BsrFI) FastDigest® BsrFI (Cfr10I) Cfr10I R↓CCGGY ER0181 105
Cfr13I (Sau96I) FastDigest® Sau96I (Cfr13I) AsuI G↓GNCC ER0191 106
Cfr42I (SacII) SacII CCGC↓GG ER0201/2/5 106CpoI (RsrII) FastDigest® RsrII (CpoI) RsrII CG↓GWCCG ER0741/2 106
CseI (HgaI) FastDigest® HgaI (CseI) HgaI GACGC(5/10)↓ ER1901 107
Csp6I (CviQI) FastDigest® Csp6I RsaI (GT↓AC) G↓TAC ER0211 107
DpnI FastDigest® DpnI DpnI Gm6A↓TC ER1701/2/5 108
DraI FastDigest® DraI AhaIII TTT↓AAA ER0221/3 108
Eam1104I (EarI) FastDigest® EarI (Eam1104I) Ksp632I CTCTTC(1/4)↓ ER0231/2 109
Eam1105I (AhdI) FastDigest® Eam1105I Eam1105I GACNNN↓NNGTC ER0241 109
Ecl136II (EcoICRI) FastDigest® Ecl136II SacI (GAGCT↓C) GAG↓CTC ER0251 109
Eco24I (BanII) HgiJII GRGCY↓C ER0281 110Eco31I (BsaI) FastDigest® Eco31I Eco31I GGTCTC(1/5)↓ ER0291/2 110
Eco32I (EcoRV) FastDigest® EcoRV (Eco32I) EcoRV GAT↓ATC ER0301/2/3/5 110
Eco47I (AvaII) FastDigest® AvaII (Eco47I) AvaII G↓GWCC ER0311/2 111
Eco47III (AfeI) FastDigest® AfeI (Eco47III) Eco47III AGC↓GCT ER0321/2 111
Eco52I (EagI) FastDigest® EagI (Eco52I) XmaIII C↓GGCCG ER0331/2 111
Eco57I (AcuI) FastDigest® AcuI (Eco57I) Eco57I CTGAAG(16/14)↓ ER0341/2 112
Eco57MI Eco57MI CTGRAG(16/14)↓ ER1671 112Eco72I (PmlI) FastDigest® PmlI (Eco72I) PmaCI CAC↓GTG ER0361 113
Eco81I (Bsu36I) FastDigest® Bsu36I (Eco81I) SauI CC↓TNAGG ER0371/2 113
Eco88I (AvaI) FastDigest® AvaI (Eco88I) AvaI C↓YCGRG ER0381 113
Eco91I (BstEII) FastDigest® Eco91I BstEII G↓GTNACC ER0391/2 114
Eco105I (SnaBI) FastDigest® SnaBI (Eco105I) SnaBI TAC↓GTA ER0401/2 114
Eco130I (StyI) FastDigest® StyI (Eco130I) StyI C↓CWWGG ER0411 114
Eco147I (StuI) FastDigest® StuI (Eco147I) StuI AGG↓CCT ER0421/2 115
EcoO109I (DraII) FastDigest® EcoO109I DraII RG↓GNCCY ER0261 115
EcoRI FastDigest® EcoRI EcoRI G↓AATTC ER0271/2/3/5 116
EcoRII EcoRII ↓CCWGG ER1921 116EheI (SfoI) FastDigest® EheI NarI (GG↓CGCC) GGC↓GCC ER0441 117
Esp3I (BsmBI) FastDigest® BsmBI (Esp3I) Esp3I CGTCTC(1/5)↓ ER0451/2 117
FaqI (BsmFI) FastDigest® BsmFI (FaqI) FinI GGGAC(10/14)↓ ER1811 118
FspAI FastDigest® FspAI FspAI RTGC↓GCAY ER1661/2 118
FspBI (BfaI) FastDigest® BfaI (FspBI) MaeI C↓TAG ER1761/2 119
GsuI (BpmI) FastDigest® BpmI (GsuI) GsuI CTGGAG(16/14)↓ ER0461/2 119
HhaI FastDigest® HhaI HhaI GCG↓C ER1851 120
Hin1I (BsaHI) FastDigest® BsaHI (Hin1I) AcyI GR↓CGYC ER0471 120
Hin1II (NlaIII ) FastDigest® NlaIII (Hin1II) NlaIII CATG↓ ER1831 120
Hin4I Hin4I ↓(8/13-14)GAY(N)5VTC(13-14/8)↓ ER1601/2 121Hin6I (HinP1I) FastDigest® HinP1I (Hin6I) HhaI (GCG↓C) G↓CGC ER0481 121
(continued on next page)
78
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
(continued on next page)
Table 1.4. Fermentas conventional restriction enzymes.
Conventional restriction enzyme
FastDigest® restriction enzyme
Prototype Specificity 5’→3’ Cat. # Page
HincII (HindII) FastDigest® HincII HindII GTY↓RAC ER0491/2 121
HindIII FastDigest® HindIII HindIII A↓AGCTT ER0501/2/3/5 122
HinfI FastDigest® HinfI HinfI G↓ANTC ER0801/2/3 122
HpaII FastDigest® HpaII HpaII C↓CGG ER0511/2 122
HphI HphI GGTGA(8/7)↓ ER1101/2 123Hpy8I (MjaIV) FastDigest® Hpy8I MjaIV GTN↓NAC ER1571/2 123
HpyF3I (DdeI) FastDigest® DdeI (HpyF3I) DdeI C↓TNAG ER1881/2 123
HpyF10VI (MwoI) FastDigest® HpyF10VI MwoI GCNNNNN↓NNGC ER1731/2 124
KpnI FastDigest® KpnI KpnI GGTAC↓C ER0521/2/3 124
Kpn2I (BspEI) FastDigest® Kpn2I BspMII T↓CCGGA ER0531/2 124
KspAI (HpaI) FastDigest® HpaI (KspAI) HpaI GTT↓AAC ER1031/2 125
LguI (SapI) FastDigest® SapI (LguI) SapI GCTCTTC(1/4)↓ ER1931/2 125
Lsp1109I (BbvI) FastDigest® BbvI (Lsp1109I) BbvI GCAGC(8/12)↓ ER2071 125
LweI (SfaNI) FastDigest® SfaNI (BmsI) SfaNI GCATC(5/9)↓ ER1621/2 126
MauBI FastDigest® MauBI MauBI CG↓CGCGCG ER2081 126
MbiI (BsrBI) FastDigest® BsrBI (MbiI) BsrBI CCGCTC(-3/-3)↓ ER1271 126
MboI FastDigest® MboI MboI ↓GATC ER0811/2 127
MboII FastDigest® MboII MboII GAAGA(8/7)↓ ER0821/2 127
MlsI (MscI) FastDigest® MscI (MlsI) BalI TGG↓CCA ER1211/2 128
MluI FastDigest® MluI MluI A↓CGCGT ER0561/2 128
MnlI FastDigest® MnlI MnlI CCTC(7/6)↓ ER1071/2 128
Mph1103I (NsiI) FastDigest® NsiI (Mph1103I) AvaIII ATGCA↓T ER0731/2 129
MreI (Sse232I) FastDigest® MreI Sse232I CG↓CCGGCG ER2021 129
MspI (HpaII) FastDigest® MspI HpaII C↓CGG ER0541/2 129
MssI (PmeI) FastDigest® MssI PmeI GTTT↓AAAC ER1341/2 130
MunI (MfeI) FastDigest® MfeI (MunI) MfeI C↓AATTG ER0751/2 130
MvaI (BstNI) FastDigest® MvaI EcoRII (↓CCWGG) CC↓WGG ER0551 130
Mva1269I (BsmI) FastDigest® Mva1269I BsmI GAATGC(1/-1)↓ ER0961/2 131
NcoI FastDigest® NcoI NcoI C↓CATGG ER0571/2/5 131
NdeI FastDigest® NdeI NdeI CA↓TATG ER0581/2/5 132
NheI FastDigest® NheI NheI G↓CTAGC ER0971/2/5 132
NmuCI (Tsp45I) FastDigest® NmuCI Tsp45I ↓GTSAC ER1511 133
NotI FastDigest® NotI NotI GC↓GGCCGC ER0591/2/3/5 133
NsbI (FspI) FastDigest® FspI (NsbI) MstI TGC↓GCA ER1221 133
OliI (AleI) FastDigest® AleI (OliI) OliI CACNN↓NNGTG ER1631/2 134
PacI FastDigest® PacI PacI TTAAT↓TAA ER2201 134
PaeI (SphI) FastDigest® SphI (PaeI) SphI GCATG↓C ER0601/2 134
PagI (BspHI) FastDigest® BspHI (PagI) BspHI T↓CATGA ER1281/2 135
PasI PasI CC↓CWGGG ER1861 135PauI (BssHII) FastDigest® BssHII (PteI) BsePI G↓CGCGC ER1091/2 135
PdiI (NaeI) FastDigest® NaeI (PdiI) NaeI GCC↓GGC ER1521/2 136
PdmI (XmnI) FastDigest® PdmI XmnI GAANN↓NNTTC ER1531/2 136
PfeI (TfiI) FastDigest® TfiI (PfeI) TfiI G↓AWTC ER1781 136
Pfl23II (BsiWI) FastDigest® BsiWI (Pfl23II) SplI C↓GTACG ER0851 137
PfoI FastDigest® PfoI PfoI T↓CCNGGA ER1751 137
PpiI PpiI ↓(7/12)GAAC(N)5CTC(13/8)↓ ER1541 138Ppu21I (BsaAI) FastDigest® BsaAI (Ppu21I) BsaAI YAC↓GTR ER1971 138
PscI (PciI) BspLU11I A↓CATGT ER1871/2 138Psp5II (PpuMI) FastDigest® PpuMI (Psp5II) PpuMI RG↓GWCCY ER0761 139
Psp1406I (AclI) FastDigest® AclI (Psp1406I) AclI AA↓CGTT ER0941/2 139
PstI FastDigest® PstI PstI CTGCA↓G ER0611/2/5 140
PsuI (BstYI) FastDigest® PsuI XhoII R↓GATCY ER1551 140
79
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.4. Fermentas conventional restriction enzymes.
Conventional restriction enzyme
FastDigest® restriction enzyme
Prototype Specificity 5’→3’ Cat. # Page
PsyI (Tth111I) FastDigest® PsyI Tth111I GACN↓NNGTC ER1331 140
PvuI FastDigest® PvuI PvuI CGAT↓CG ER0621/2 141
PvuII FastDigest® PvuII PvuII CAG↓CTG ER0631/3/5 141
RsaI FastDigest® RsaI RsaI GT↓AC ER1121/2 141
RseI (MslI) FastDigest® MslI (RseI) MslI CAYNN↓NNRTG ER2001/2 142
SacI FastDigest® SacI SacI GAGCT↓C ER1131/2/5 142
SalI FastDigest® SalI SalI G↓TCGAC ER0641/2/5 143
SatI (Fnu4HI) FastDigest® Fnu4HI (SatI) Fnu4HI GC↓NGC ER1641/2 143
ScaI FastDigest® ScaI ScaI AGT↓ACT ER0431/2 144
SchI (MlyI) FastDigest® MlyI (SchI) PleI (GAGTC(4/5)↓) GAGTC(5/5)↓ ER1371 144
SdaI (SbfI) FastDigest® SbfI (SdaI) Sse8387I CCTGCA↓GG ER1191/2 144
SduI (Bsp1286I) FastDigest® Bsp1286I (SduI) SduI GDGCH↓C ER0651 145
SfaAI (AsiSI) FastDigest® AsiSI (SfaAI) SgfI GCGAT↓CGC ER2091 145
SfiI FastDigest® SfiI SfiI GGCCNNNN↓NGGCC ER1821 146
SgeI SgeI m5CNNG(9/13)↓ ER2211 146SgrDI SgrDI CG↓TCGACG ER2031 147SgsI (AscI) FastDigest® AscI (SgsI) AscI GG↓CGCGCC ER1891/2 147
SmaI FastDigest® SmaI SmaI CCC↓GGG ER0661/2/3/5 147
SmiI (SwaI) FastDigest® SwaI (SmiI) SwaI ATTT↓AAAT ER1241 148
SmoI (SmlI) SmlI C↓TYRAG ER1981 148SmuI (FauI) FauI CCCGC(4/6)↓ ER1691 148SsiI (AciI) FastDigest® AciI (SsiI) AciI CCGC(-3/-1)↓ ER1791 149
SspI FastDigest® SspI SspI AAT↓ATT ER0771/2 149
SspDI (KasI) NarI (GG↓CGCC) G↓GCGCC ER2191 150TaaI (HpyCH4III) FastDigest® TaaI Tsp4CI ACN↓GT ER1361/2 150
TaiI (MaeII) FastDigest® TaiI MaeII (A↓CGT) ACGT↓ ER1141/2 150
TaqI FastDigest® TaqI TaqI T↓CGA ER0671/2 151
TasI (Tsp509I) FastDigest® Tsp509I (TasI) TspEI ↓AATT ER1351/2 151
TatI FastDigest® TatI TatI W↓GTACW ER1291 151
TauI FastDigest® TauI TauI GCSG↓C ER1651/2 152
Tru1I (MseI) FastDigest® Tru1I MseI T↓TAA ER0981/2/3 152
TscAI (TspRI) FastDigest® TspRI (TscAI) TspRI CASTG(2/-7)↓ ER2101 153
TsoI TsoI TARCCA(11/9)↓ ER1991 153TstI TstI ↓(8/13)CAC(N)6TCC(12/7)↓ ER1911 154Van91I (PflMI) FastDigest® PflMI (Van91I) PflMI CCANNNN↓NTGG ER0711/2 154
VspI (AseI) FastDigest® AseI (VspI) VspI AT↓TAAT ER0911/2 154
XagI (EcoNI) FastDigest® EcoNI (XagI) EcoNI CCTNN↓NNNAGG ER1301 155
XapI (ApoI) FastDigest® XapI ApoI R↓AATTY ER1381 155
XbaI FastDigest® XbaI XbaI T↓CTAGA ER0681/2/3/5 155
XceI (NspI) FastDigest® NspI (XceI) NspI RCATG↓Y ER1471/2 156
XhoI FastDigest® XhoI XhoI C↓TCGAG ER0691/2/3/5 156
XmaJI (AvrII) FastDigest® AvrII (XmaJI) AvrII C↓CTAGG ER1561/2 156
XmiI (AccI) FastDigest® AccI (XmiI) AccI GT↓MKAC ER1481/2 157
Nicking enzymes
Nb.Bpu10I Nb.Bpu10ICCTNA GC GGANT↑CG
ER1681 157
Nt.Bpu10I Nt.Bpu10ICC↓TNAGCGG ANTCG
ER2011 158
Nb.Mva1269I Nb.BsmIGAATG CCTTAC↑G
ER2051 158
Homing enzyme
I-SceI I-SceITAGGG ATAA↓CAGGGTAATATCCC↑TATT GTCCCATTA
ER1771 159
Alphabetic list of commercially available restriction enzymes see p.207.
80
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Product Description
5’...T T A↓T A A ...3’
3’...A A T↑A T T ...5’
#ER2061 200 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® PsiI (AanI)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferAanI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with AanI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
AanI (PsiI) B G O R Tango 2X Tango 50-100 50-100 0-20 0-20 100 20-50
Activity in Five Buffer System, %
5’...C A C C T G C (N)4↓ ...3’
3’...G T G G A C G (N)8↑ ...5’
#ER1581 25 uSupplied with:10X Buffer AarI 1 ml10X Buffer Tango™ 1 ml50X oligonucleotide 25µl
#ER1582 125 uSupplied with:10X Buffer AarI 1 ml10X Buffer Tango™ 1 ml50X oligonucleotide 2x25 µl
Concentration2 u/µl
Conditions for 100% Activity 1X Buffer AarI at 37°C.Oligonucleotide 0.5 µM.
Storage BufferAarI is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with AarI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for digestion of 1 µg of agarose-embedded λ DNA in 16 hours.
AarI B G O R Tango 2X Tango NR NR 0-20 0-20 NR 50-100
Activity in Five Buffer System, %
Note• For cleavage with AarI at least two copies of
its recognition sequence are required.• Inclusion of 0.5 µM oligonucleotide with the
AarI recognition sequence in the reaction mixture significantly improves cleavage of
DNAs, especially of those with a single AarI site. Still, a complete cleavage of some substrates with AarI is difficult to achieve.
• Greater than 10-fold overdigestion with AarI may result in star activity.
• AarI may remain associated with the cleaved
DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
5’...G A C N N N N↓N N G T C ...3’
3’...C T G N N↑N N N N C A G ...5’
#ER1721 300 uSupplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® DrdI (AasI)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer B at 37°C.
Storage BufferAasI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with AasI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1µg of agarose-embed-ded λ DNA in 16 hours.
NoteGreater than 10-fold overdigestion with AasI may result in star activity.
* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
AasI (DrdI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 20-50 0-20 0-20 50-100* 0-20
81
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...G↓ G T A C C...3’
3’...C C A T G↑ G...5’
#ER0901 1000 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
#ER0902 5000 uSupplied with:10X Buffer O 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® Acc65I
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer O at 37°C.
Storage BufferAcc65I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Acc65I, approximately 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Acc65I (Asp718I) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 20-50 20-50 50-100
NoteAcc65I cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
AccI Fermentas enzymes FastDigest® AccI (XmiI) and XmiI (AccI)
AccII Fermentas enzymes FastDigest® Bsh1236I and Bsh1236I (BstUI)
AccIII Fermentas enzymes FastDigest® Kpn2I and Kpn2I (BspEI) (different sensitivity to methylation)
AccB7I Fermentas enzymes FastDigest® PflMI (Van91I) and Van91I (PflMI)
AciI Fermentas enzymes FastDigest® AciI (SsiI) and SsiI (AciI)
AclI Fermentas enzymes FastDigest® AcII (Psp1406I) and Psp1406I (AclI)
AcsI Fermentas enzymes FastDigest® XapI and XapI (ApoI)
AcuI Fermentas enzymes FastDigest® AcuI (Eco57I) and Eco57I (AcuI)
AcyI Fermentas enzymes FastDigest® BsaHI (Hin1I) and Hin1I (BsaHI)
5’...G A C G T↓ C...3’
3’...C↑ T G C A G...5’
#ER0991 300 u#ER0992 1500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® AatII
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferAatII is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with AatII, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1µg of agarose-embed-ded λ DNA in 16 hours.
NoteActivity decreases if reaction buffer pH is not between 7.5 and 8.0 at 25°C.
AatII Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 0-20 0-20 100 20-50
82
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
AdeI (DraIII) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 100 20-50 100 100* 20-50* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
5’...C A C N N N↓G T G...3’
3’...G T G↑N N N C A C...5’
#ER1231 500 uSupplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® DraIII (AdeI)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer G at 37°C.
Storage BufferAdeI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with AdeI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1µg of agarose-embedded λ DNA in 16 hours.
NoteGreater than 20-fold overdigestion with AdeI may result in star activity.
5’...C A C↓G T C...3’
3’...G T G↑C A G...5’
#ER1941 200 uSupplied with:10X Buffer AjiI 1 ml10X Buffer Tango™ 1 ml
Concentration5 u/µl
Conditions for 100% Activity 1X Buffer AjiI at 37°C.
Storage BufferAjiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with AjiI, approxi-mately 80% of the DNA fragments can be ligated. No more than 50% of these can be recut due to the asymmetric recognition sequence of AjiI. The remaining uncleaved ligation products may be cut by AatII and Eco72I (PmlI).
Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
AjiI (BmgBI) B G O R Tango 2X Tango NR NR 20-50* NR NR 20-50** Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
Activity in Five Buffer System, %
NoteLow salt, high glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.
AfaI Fermentas enzymes FastDigest® Csp6I and Csp6I (CviQI) (different cleavage position); FastDigest® RsaI and RsaI
AfeI Fermentas enzymes FastDigest® AfeI (Eco47III) and Eco47III (AfeI)
AflII Fermentas enzymes FastDigest® AflII (BspTI) and BspTI (AflII)
AgeI Fermentas enzymes FastDigest® AgeI (BshTI) and BshTI (AgeI)
AhaIII Fermentas enzymes FastDigest® DraI and DraI
AhdI Fermentas enzymes FastDigest® Eam1105I and Eam1105I (AhdI)
83
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
AleI Fermentas enzymes FastDigest® AleI (OliI) and OliI (AleI)
5’...↓10(N)GCA(N)6TGC(N)12↓ ...3’
3’...↑12(N)CGT(N)6ACG(N)10↑...5’
#ER1801 50 uSupplied with:10X Buffer R 1 ml50X SAM 0.1 ml10X BufferTango™ 1 ml
Concentration2 u/µl
Conditions for 100% Activity1X Buffer R at 37°C.SAM 0.01 mM.
Storage BufferAlfI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 20-fold overdigestion with AlfI, approximately 70% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1µg of agarose-embed-ded λ DNA in 16 hours.
AlfI Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM
0-20 0-20 0-20 100 0-20 20-50
Note• AlfI requires S-adenosylmethionine for activity.• Complete cleavage of some substrates with
AlfI is difficult to achieve.• AlfI concentration is determined by the
maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.
5’...↓ 7(N)GAA(N)7TTGG(N)11 ↓ ...3’
3’...↑12(N)CTT(N)7AACC(N)6 ↑...5’
#ER1951 100 uSupplied with:10X Buffer R 1 ml50X SAM 0.1 ml10X BufferTango™ 1 ml
Also available asFastDigest® AjuI
Concentration5 u/µl
Conditions for 100% Activity1X Buffer R at 37°C.SAM 0.01 mM.
Storage BufferAjuI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with AjuI, more than 90% of the DNA fragments can be ligated, but none of these can be recut due to the methyla-tion of the recognition sequence by this enzyme.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
AjuI Activity in Five Buffer System, %
Note• AjuI requires S-adenosylmethionine for activity.• Complete cleavage of some substrates with
AjuI is difficult to achieve.• AjuI concentration is determined by the
maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.
• AjuI produces double-strand cuts on both sides of the interrupted recognition site. In certain sequence contexts, the cleavage position may be shifted by one base pair. However, the cleavage position indicated above will predominate.
B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM
0-20 50-100 20-50 100 50-100 50-100
84
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
AlwI Fermentas enzyme BspPI (AlwI)
5’...↓ 7(N)GAAC(N)6TCC(N)12-13↓...3’
3’...↑12-13(N)CTTG(N)6AGG(N)7 ↑...5’
#ER1491 100 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Concentration2 u/µl
Conditions for 100% Activity1X Buffer R at 30°C.
Storage BufferAloI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with AloI, approxi-mately 70% of the DNA fragments can be ligated and more than 80% of these can be recut.
Methylation EffectsDam: may overlap – effect not determined.Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
AloI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 100 20-50 100
Note• Incubation at 37°C results in 20% activity.• AloI produces double-strand cuts on both
sides from the interrupted recognition site. Its unique feature is a degenerate cleavage point on the 3’ side of the recognition sequence (12 or 13 nt away).
• The presence of S-adenosylmethionine in the reaction mixture results in incomplete cleavage with AloI.
• Greater than 10-fold overdigestion with AloI may result in star activity.
• AloI may remain associated with the cleaved DNA. This may cause DNA band shifting
during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
5’...G W G C W↓C...3’
3’...C↑W C G W G...5’
#ER0021 500 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® Alw21I
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer O at 37°C.
Storage BufferAlw21I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Alw21I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Alw21I (BsiHKAI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 50-100 20-50 50-100
5’...A G↓C T...3’
3’...T C↑G A...5’
#ER0011 600 uSupplied with:10X Buffer Tango™ 1 ml
#ER0012 3000 uSupplied with:10X Buffer Tango™ 2x1 ml
Also available asFastDigest® AluI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferAluI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.15% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with AluI, approxi-mately 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – blocked (p.181).
AluI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 0-20 0-20 0-20 100 20-50
85
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...G T C T C(N)1↓...3’
3’...C A G A G(N)5↑...5’
#ER0031 1000 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® Alw26I
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferAlw26I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Alw26I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
Alw26I (BsmAI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 100 0-20 0-20 100 100
5’...G↓T G C A C...3’
3’...C A C G T↑G...5’
#ER0041 1000 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® ApaLI (Alw44I)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferAlw44I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Alw44I, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – blocked (p.179).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Alw44I (ApaLI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 100 0-20 50-100 100 50-100
ApaI Activity in Five Buffer System, % B G O R Tango 2X Tango 100 20-50 0-20 0-20 20-50 0-20
5’...G G G C C↓C...3’
3’...C↑C C G G G...5’
#ER1411 3000 u#ER1415 5000 u
Both supplied with:10X Buffer B 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® ApaI
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer B at 37°C.
Storage Buffer ApaI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with ApaI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Note• Incubation at 30°C results in a 2-fold
increase in activity.• ApaI is inhibited by salt concentrations
above 50 mM.• ApaI cleavage is impaired by overlapping
dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
AlwNI Fermentas enzymes FastDigest® AIwNI (CaiI) and CaiI (AlwNI)
Aor13HI Fermentas enzymes FastDigest® Kpn2I and Kpn2I (BspEI)
Aor51HI Fermentas enzymes FastDigest® AfeI (Eco47III) and Eco47III (AfeI)
86
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
BamHI Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50* 100 20-50 50-100* 100* 50-100* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
5’...G↓G A T C C...3’
3’...C C T A G↑G...5’
#ER0051 4000 uSupplied with:10X Buffer BamHI 2x1 ml10X Buffer Tango™ 1 ml
#ER0055 10000 uSupplied with:10X Buffer BamHI 4x1 ml10X Buffer Tango™ 1 ml
#ER0052 5x4000 u#ER0053 HC, 20000 u
Both supplied with:10X Buffer BamHI 4x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BamHI
Concentration10 u/µl50 u/µl, HC
Conditions for 100% Activity 1X Buffer BamHI at 37°C.
Storage BufferBamHI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 200 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.15% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BamHI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NoteLow salt, high glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.
ApaLI Fermentas enzymes FastDigest® ApaLI (Alw44I) and Alw44I (ApaLI) (different sensitivity to methylation)
ApoI Fermentas enzymes FastDigest® XapI and XapI (ApoI)
AscI Fermentas enzymes FastDigest® AscI (SgsI) and SgsI (AscI)
AseI Fermentas enzymes FastDigest® AseI (VspI) and VspI (AseI)
AsiSI Fermentas enzymes FastDigest® AsiSI (SfaAI) and SfaAI (AsiSI)
AspI Fermentas enzymes FastDigest® PsyI and PsyI (Tth111I)
Asp700I Fermentas enzymes FastDigest® PdmI and PdmI (XmnI)
Asp718I Fermentas enzymes FastDigest® Acc65I and Acc65I (Asp718I); FastDigest® KpnI and KpnI (different cleavage position and different sensitivity to methylation)
AspEI Fermentas enzymes FastDigest® Eam1105I and Eam1105I (AhdI)
AspHI Fermentas enzymes FastDigest® Alw21I and Alw21I (BsiHKAI)
AsuI Fermentas enzymes FastDigest® Sau96I (Cfr13I) and Cfr13I (Sau96I)
AsuII Fermentas enzymes FastDigest® Bsp119I and Bsp119I (BstBI)
AvaI Fermentas enzymes FastDigest® AvaI (Eco88I) and Eco88I (AvaI)
AvaII Fermentas enzymes FastDigest® AvaII (Eco47I) and Eco47I (AvaII)
AvaIII Fermentas enzymes FastDigest® NsiI (Mph1103I) and Mph1103I (NsiI)
AviII Fermentas enzymes FastDigest® FspI (NsbI) and NsbI (FspI)
AvrII Fermentas enzymes FastDigest® AvrII (XmaJI) and XmaJI (AvrII)
BaeGI Fermentas enzymes FastDigest® Bme1580I (BseSI) and BseSI (Bme1580I)
BalI Fermentas enzymes FastDigest® MscI (MlsI) and MlsI (MscI)
BanI Fermentas enzymes FastDigest® BanI (BshNI) and BshNI (BanI)
BanII Fermentas enzyme Eco24I (BanII)
87
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...C↓A C G A G...3’
3’...G T G C T↑C...5’
#ER1841 200 uSupplied with:10X Buffer Tango™ 1 ml
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferBauI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.15% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BauI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG – no effect.EcoKI, EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1µg of agarose-embed-ded λ DNA in 16 hours.
BauI (BssSI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 0-20 50-100 100 50-100
BclI Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 20-50 20-50 100* 100* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
5’...T↓G A T C A...3’
3’...A C T A G↑T...5’
#ER0721 1000 uSupplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml
#ER0725 3000 u#ER0722 5000 u
Both supplied with:10X Buffer G 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BclI
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer G at 55°C.
Storage BufferBclI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with BclI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam: completely overlaps – blocked (p.176).Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – blocked (p.181).
Note• Incubation at 37°C results in 50% activity.• Greater than 15-fold overdigestion with BclI
may result in star activity.• BclI is blocked by dam methylation. To avoid
dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
BbeI Fermentas enzymes FastDigest® EheI and EheI (SfoI) (different cleavage position); Fermentas enzyme SspDI (KasI) (different cleavage position)
BbrPI Fermentas enzymes FastDigest® PmlI (Eco72I) and Eco72I (PmlI)
BbsI Fermentas enzymes FastDigest® BbsI (BpiI) and BpiI (BbsI)
BbuI Fermentas enzymes FastDigest® SphI (PaeI) and PaeI (SphI)
BbvI Fermentas enzymes FastDigest® BbvI (Lsp1109I) and Lsp1109I (BbvI); FastDigest® BseXI and BseXI (BbvI)
BbvII Fermentas enzymes FastDigest® BbsI (BpiI) and BpiI (BbsI)
BciVI Fermentas enzyme BfuI (BciVI)
88
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...C C↓S G G...3’
3’...G G S↑C C...5’
#ER0061 1000 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® NciI (BcnI)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferBcnI is supplied in: 10 mM potassium phosphate (pH 7.5 at 25°C), 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with BcnI, more than 80% and more than 95% of these can be recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – cleavage impaired (p.178).
BcnI (NciI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 50-100 50-100 100 50-100
5’...A↓C T A G T...3’
3’...T G A T C↑A...5’
#ER1251 400 u#ER1252 2000 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® SpeI (BcuI)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage Buffer BcuI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BcuI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm, CpG – no effect.EcoKI, EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded Adenovirus-2 DNA in 16 hours.
BcuI (SpeI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 20-50 100 0-20
5’...↓10(N)TGA(N)6TCA(N)12↓ ...3’
3’...↑12(N)ACT(N)6AGT(N)10↑...5’
#ER1961 50 uSupplied with:10X Buffer G 1 ml50X SAM 0.1 ml10X BufferTango™ 1 ml
Concentration2 u/µl
Conditions for 100% Activity1X Buffer G at 30°C.SAM 0.05 mM.
Storage BufferBdaI is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with BdaI, more than 70% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.
Methylation EffectsDam: may overlap – blocked (p.176).Dcm, CpG, EcoKI, EcoBI – no effect.
BdaI Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM
NR 100 0-20 20-50 50-100* 50-100* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
Note• Incubation at 37°C results in 30% activity.• Requires S-adenosylmethionine for activity.
Complete cleavage of some substrates with BdaI is difficult to achieve.
• BdaI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.
• BdaI produces double-strand cuts on both sides of the interrupted recognition site. In certain sequence contexts, the cleavage position may be shifted by one base pair. However, the cleavage position indicated above will predominate.
• Greater than 40-fold overdigestion with BdaI may result in star activity.
• BdaI may remain associated with the cleaved
DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample prepara-tion or heat the digested DNA in the presence of SDS prior to electrophoresis.
• BdaI is blocked by overlapping dam methy-lation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
89
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...C↓T R Y A G...3’
3’...G A Y R T↑C...5’
#ER1161 200 u#ER1162 1000 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® SfcI (BfmI)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage Buffer BfmI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BfmI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
BfmI (SfcI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 0-20 0-20 100 20-50
NoteLow salt, high glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.
BfaI Fermentas enzymes FastDigest® BfaI (FspBI) and FspBI (BfaI)
5’...A C T G G G(N)5↓...3’
3’...T G A C C C(N)4↑...5’
#ER1591 50 uSupplied with:10X Buffer Tango™ 1 ml3X BfiI Stop Solution 0.5 ml
Concentration2 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
BfiI (BmrI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 0-20 0-20 100 0-20
Note• BfiI restriction enzyme cleaves DNA specifically
both in the presence and absence of Mg2+ ions in the reaction mixture (Sapranauskas, R., et al., J.Biol.Chem., 275, 30878-30885, 2000).
• Chelating of Mg2+ ions by EDTA does not inhibit BfiI activity and may cause non-specific products. The non-specific cleavage increases at temperatures over 37°C.
• Greater than 40-fold overdigestion with BfiI may result in star activity.
Termination of Digestion ReactionDo not attempt to stop the digestion reaction by adding EDTA (see Note). Use Stop Solution containing SDS or the supplied BfiI Stop Solu-tion to terminate the reaction by incubating at 65°C for 10 min. If the digested DNA is to be used in down-stream manipulations, inactivate BfiI by incubating at 65°C for 20 min.
3X BfiI Stop Solution (supplied)0.6% SDS, 0.05% bromophenol blue and 50% glycerol.
Storage BufferBfiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with BfiI, more than 40% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm, CpG – no effect.EcoKI, EcoBI: may overlap – effect not determined.
BfrI Fermentas enzymes FastDigest® AflII (BspTI) and BspTI (AflII)
BfrBI Fermentas enzymes FastDigest® NsiI (Mph1103I) and Mph1103I (NsiI) (different cleavage position)
90
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...G T A T C C(N)6↓...3’
3’...C A T A G G(N)5↑...5’
#ER1501 100 uSupplied with:10X Buffer BfuI 1 ml10X Buffer Tango™ 1 ml
Concentration5 u/µl
Conditions for 100% Activity 1X Buffer BfuI at 37°C.
Storage Buffer BfuI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 300 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 5-fold overdigestion with BfuI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI, CpG – no effect.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1µg of agarose-embed-ded λ DNA in 16 hours.
BfuI (BciVI) B G O R Tango 2X Tango NR NR 0-20 0-20 NR 50-100** Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
Activity in Five Buffer System, %
NoteGreater than 5-fold overdigestion with BfuI may result in star activity.
BfuAI Fermentas enzymes FastDigest® BspMI (BveI) and BveI (BspMI)
BfuCI Fermentas enzymes FastDigest® Sau3AI (Bsp143I) and Bsp143I (Sau3AI); FastDigest® DpnI and DpnI (different cleavage position and different sensitivity to methylation); FastDigest® MboI and MboI (different sensitivity to methylation)
5’...G C C N N N N↓N G G C...3’
3’...C G G N↑N N N N C C G...5’
#ER0071 2000 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
#ER0072 5x2000 uSupplied with:10X Buffer O 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BglI
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer O at 37°C.
Storage BufferBglI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BglI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
BglI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 100 100 0-20 100
5’...A↓G A T C T...3’
3’...T C T A G↑A...5’
#ER0081 500 u#ER0082 2500 u
Both supplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BglII
Concentration10 u/µl
Conditions for 100% Activity1X Buffer O at 37°C.
Storage BufferBglII is supplied in: 10 mM Tris-HCl (pH 8.2 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BglII, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – cleavage impaired (p.181).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
BglII Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 50-100 0-20 100
91
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...G A C N N↓N N G T C...3’
3’...C T G N N↑N N C A G...5’
#ER1431 500 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® PshAI (BoxI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferBoxI is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.15% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BoxI, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded λ DNA in 16 hours.
BoxI (PshAI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 20-50 100 20-50
NoteIncubation at 30°C results in a 2.5-fold increase in activity.
5’...C C↓N G G...3’
3’...G G N↑C C...5’
#ER1421 500 u#ER1422 2500 u
Both supplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® ScrFI (Bme1390I)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer O at 37°C.
Storage BufferBme1390I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Bme1390I, more than 80% of the DNA fragments can be ligate-dand more than 90% of these can be recut.Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG may overlap – blocked (p.177, 179).
Bme1390I (ScrFI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 100 50-100 50-100 50-100
NoteBme1390I is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
BinI Fermentas enzyme BspPI (AlwI)
BlnI Fermentas enzymes FastDigest® AvrII (XmaJI) and XmaJI (AvrII)
BlpI Fermentas enzymes FastDigest® BlpI (Bpu1102I) and Bpu1102I (BlpI)
Bme1580I Fermentas enzymes FastDigest® Bme1580I (BseSI) and BseSI (Bme1580I)
BmgBI Fermentas enzyme AjiI (BmgBI)
BmrI Fermentas enzyme BfiI (BmrI)
BmtI Fermentas enzymes FastDigest® BmtI (BspOI) and BspOI (BmtI); FastDigest® NheI and NheI (different cleavage position)
BmyI Fermentas enzymes FastDigest® Bsp1286I (SduI) and SduI (Bsp1286I)
92
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
BpiI (BbsI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 50-100 50-100 50-100 50-100
5’...G A A G A C(N)2↓...3’
3’...C T T C T G(N)6↑...5’
#ER1011 200 u#ER1012 1000 u
Both supplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BbsI (BpiI)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer G at 37°C.
Storage BufferBpiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BpiI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
BpmI Fermentas enzymes FastDigest® BpmI (GsuI) and GsuI (BpmI)
5’...↓ 8(N)G A G(N)5C T C(N)13↓...3’
3’...↑13(N)C T C(N)5G A G(N) 8↑...5’
#ER1311 100 uSupplied with:10X Buffer Tango™ 1 ml50X SAM 0.1 ml
#ER1312 500 uSupplied with:10X Buffer Tango™ 1 ml50X SAM 2x0.1 ml
Also available asFastDigest® BpII
Concentration5 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.SAM 0.05 mM.
Storage BufferBplI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with BplI, more than 70% of the DNA fragments can be ligated, but none of these can be recut due to the methyla-tion of the recognition sequence by this enzyme.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded λ DNA in 16 hours.
Note• BplI requires only Mg2+ for its activity, but
is stimulated by S-adenosylmethionine. 0.05 mM S-adenosylmethionine gives more than a 100-fold increase in BplI activity. Still, complete cleavage of some substrates with BplI is difficult to achieve.
• BplI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.
BplI Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM
0-20 20-50 0-20 0-20 100 20-50
93
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
BpuAI Fermentas enzymes FastDigest® BbsI (BpiI) and BpiI (BbsI)
BsaI Fermentas enzymes FastDigest® Eco31I and Eco31I (BsaI)
BsaAI Fermentas enzymes FastDigest® BsaAI (Ppu21I) and Ppu21I (BsaAI)
BsaBI Fermentas enzymes FastDigest® BsaBI (BseJI) and BseJI (BsaBI)
BsaHI Fermentas enzymes FastDigest® BsaHI (Hin1I) and Hin1I (BsaHI)
BsaJI Fermentas enzymes FastDigest® BsaJI (BseDI) and BseDI (BsaJI)
BsaMI Fermentas enzymes FastDigest® Mva1269I and Mva1269I (BsmI)
BseAI Fermentas enzymes FastDigest® Kpn2I and Kpn2I (BspEI)
5’...G C↓T N A G C...3’
3’...C G A N T↑C G...5’
#ER0091 200 u#ER0092 1000 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® BlpI (Bpu1102I)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferBpu1102I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Bpu1102I, more than 80% of the DNA fragments can be ligated. More than 95% of these can be recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Bpu1102I (BlpI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 20-50 100 20-50
5’...C C↓T N A G C...3’
3’...G G A N T↑C G...5’
#ER1181 200 uSupplied with:10X Buffer Bpu10I 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® Bpu10I
Concentration5 u/µl
Conditions for 100% Activity 1X Buffer Bpu10I at 37°C.
Storage BufferBpu10I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Bpu10I, more than 90% of the DNA fragments can be ligated. No more than 50% of these can be recut due to asymmetric recognition sequence of Bpu10I.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Note• For cleavage with Bpu10I at least two copies
of its recognition sequence are required.• A large excess of enzyme (>4 u/1 µg DNA)
may result in incomplete DNA cleavage. There-fore, we recommend increasing the incubation time instead of using an excess of Bpu10I.
• Low salt, high glycerol (>5%) concentra-tions, pH >7.0 or a large excess of enzyme may result in star activity.
Bpu10I Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50* 50-100* 100* 50-100* 100** Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
94
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
BseDI (BsaJI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 0-20 0-20 100 50-100
5’...C↓C N N G G...3’
3’...G G N N C↑C...5’
#ER1081 300 u#ER1082 1500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® BsaJI (BseDI)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 55°C.
Storage Buffer BseDI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BseDI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
NoteIncubation at 37°C results in 10% activity.
5’...G G A T G N N↓...3’
3’...C C T A C↑N N ...5’
#ER0871 500 u#ER0872 2500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® BseGI
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 55°C.
Storage Buffer BseGI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BseGI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
BseGI (BtsCI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 20-50 20-50 100 20-50
NoteIncubation at 37°C results in 25% activity.
5’...G A T N N↓N N A T C...3’
3’...C T A N N↑N N T A G...5’
#ER1711 2000 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BsaBI (BseJI)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer O at 65°C.
Storage Buffer BseJI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with BseJI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam: may overlap – blocked (p.176).Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Note• Incubation at 37°C results in less than 10%
activity.• Greater than 20-fold overdigestion with
BseJI may result in star activity.• BseJI is blocked by overlapping dam
methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
BseJI (BsaBI) Activity in Five Buffer System, % B G O R Tango 2X Tango NR 100* 100 NR NR 100** Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
95
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
BseLI (BslI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 50-100 20-50 100 50-100
5’...C C N N N N N↓N N G G...3’
3’...G G N N↑N N N N N C C...5’
#ER1201 500 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® BslI (BseLI)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 55°C.
Storage BufferBseLI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BseLI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179).
Note• Incubation at 37°C results in 40% activity.• BseLI cleavage is impaired by overlapping dcm
methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
BseMI (BsrDI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 0-20 100 50-100 50-100
5’...G C A A T G N N↓...3’
3’...C G T T A C↑N N ...5’
#ER1261 100 u#ER1262 500 u
Both supplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BsrDI (BseMI)
Concentration5 u/µl
Conditions for 100% Activity 1X Buffer R at 55°C.
Storage BufferBseMI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BseMI, more than 95% of the DNA fragments can be ligated and approximately 80% of these can be recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
NoteIncubation at 37°C results in 20% activity.
5’...C T C A G(N)10↓...3’
3’...G A G T C(N) 8↑...5’
#ER1401 100 uSupplied with:10X Buffer Tango™ 1 ml50X SAM 0.1 ml
Also available asFastDigest® BspCNI (BseMII)
Concentration1 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 55°C.SAM 0.01 mM.
Storage BufferBseMII is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with BseMII, more than 90% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme (see Note).
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Note• Incubation at 37°C results in 30% activity.• Requires S-adenosylmethionine for activity.
Sinefungin can replace SAM in the restriction reaction. In this case DNA is not methylated and more than 95% of the ligated BseMII fragments can be recut by this enzyme.
BseMII (BspCNI) Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM
50-100 50-100 50-100 50-100 100 50-100
96
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
BseNI (BsrI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 20-50 0-20 0-20 50-100 20-50
5’...A C T G G N↓...3’
3’...T G A C↑C N ...5’
#ER0881 1000 uSupplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
#ER0882 5000 uSupplied with:10X Buffer B 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BseNI
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer B at 65°C.
Storage Buffer BseNI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BseNI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG – no effect.EcoKI, EcoBI: may overlap – effect not determined.
NoteIncubation at 37°C results in less than 10% activity.
BsePI Fermentas enzymes FastDigest® BssHII (PteI) and PauI (BssHII)
BseSI (Bme1580I) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 0-20 20-50 50-100 0-20
5’...G K G C M↓C...3’
3’...C↑M C G K G...5’
#ER1441 500 uSupplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® Bme1580I (BseSI)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer G at 55°C.
Storage BufferBseSI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BseSI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – cleavage impaired (p.181).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Note• Incubation at 37°C results in 20% activity.• Low salt, high glycerol (>5%) concentra-
tions, pH >8.0 or a large excess of enzyme may result in star activity.
5’...G C A G C(N) 8↓...3’
3’...C G T C G(N)12↑...5’
#ER1451 100 u#ER1452 500 u
Both supplied with:10X Buffer BseXI 1 ml
Also available asFastDigest® BseXI
Concentration3 u/µl
Conditions for 100% Activity 1X Buffer BseXI at 65°C.
Storage BufferBseXI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with BseXI, more than 95% of the DNA fragments can be ligated recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Note• Incubation at 37°C results in 10% activity.• Greater than 40-fold overdigestion with
BseXI may result in star activity.• BseXI may remain associated with the cleaved
DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
BseXI (BbvI) Activity in Five Buffer System, % B G O R Tango 2X Tango NR NR NR NR NR NR
97
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
BshNI (BanI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 50-100 0-20 100
5’...G↓G Y R C C...3’
3’...C C R Y G↑G...5’
#ER1001 2000 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BanI (BshNI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer O at 37°C.
Storage BufferBshNI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BshNI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, EcoBI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179).EcoKI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NoteBshNI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
5’...C G↓C G...3’
3’...G C↑G C...5’
#ER0921 500 u#ER0922 2500 u
Both supplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® Bsh1236I
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer R at 37°C.
Storage BufferBsh1236I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Bsh1236I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Bsh1236I (BstUI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 50-100 100 20-50 50-100
BseYI Fermentas enzyme FastDigest® PspFI (different cleavage position)
5’...C G R Y↓C G...3’
3’...G C↑Y R G C...5’
#ER0891 600 uSupplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BsiEI (Bsh1285I)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer G at 37°C.
Storage BufferBsh1285I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 150 mM KCl, 1 mM DTT, 1 mM EDTA, 0.15% Triton X-100, 0.5 mg ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Bsh1285I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam: may overlap – cleavage impaired (p.176).Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Bsh1285I (BsiEI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 20-50 0-20 0-20 20-50
NoteBsh1285I cleavage is impaired by overlapping dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
98
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
BshTI (AgeI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 50-100 20-50 20-50
5’...A↓C C G G T...3’
3’...T G G C C↑A...5’
#ER1461 200 u#ER1462 1000 u
Both supplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® AgeI (BshTI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer O at 37°C.
Storage BufferBshTI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BshTI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 20 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded λ DNA in 16 hours.
NoteLow salt, high glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.
BsiEI Fermentas enzymes FastDigest® BsiEI (Bsh1285I) and Bsh1285I (BsiEI)
BsiHKAI Fermentas enzymes FastDigest® Alw21I and Alw21I (BsiHKAI)
BsiWI Fermentas enzymes FastDigest® BsiWI (Pfl23II) and Pfl23II (BsiWI)
BsiYI Fermentas enzymes FastDigest® BslI (BseLI) and BseLI (BslI)
BslI Fermentas enzymes FastDigest® BslI (BseLI) and BseLI (BslI)
BsmI Fermentas enzymes FastDigest® Mva1269I and Mva1269I (BsmI)
BsmAI Fermentas enzymes FastDigest® Alw26I and Alw26I (BsmAI)
BsmBI Fermentas enzymes FastDigest® BsmBI (Esp3I) and Esp3I (BsmBI)
BsmFI Fermentas enzymes FastDigest® BsmFI (FaqI) and FaqI (BsmFI)
BsoBI Fermentas enzymes FastDigest® AvaI (Eco88I) and Eco88I (AvaI) (different sensitivity to methylation)
5’...T C G↓C G A...3’
3’...A G C↑G C T...5’
#ER0111 800 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® NruI (RruI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer O at 37°C.
Storage BufferBsp68I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with Bsp68I, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NoteGreater than 15-fold overdigestion with Bsp68I may result in star activity.
Bsp68I (NruI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 50-100 20-50 50-100
99
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
Bsp1286I Fermentas enzymes FastDigest® Bsp1286I (SduI) and SduI (Bsp1286I)
5’...T T↓C G A A...3’
3’...A A G C↑T T...5’
#ER0121 2500 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® Bsp119I
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferBsp119I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Bsp119I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).EcoKI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Bsp119I (BstBI) B G O R Tango 2X Tango 20-50 0-20 0-20 0-20 100 100
Activity in Five Buffer System, %
Bsp120I (PspOMI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 20-50 0-20 20-50 50-100 0-20
5’...G↓G G C C C...3’
3’...C C C G G↑G...5’
#ER0131 1500 uSupplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® Bsp120I
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer B at 37°C.
Storage BufferBsp120I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Bsp120I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – blocked (p.177, 179).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NoteBsp120I is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
5’...↓G A T C ...3’
3’... C T A G↑...5’
#ER0781 300 u#ER0782 1500 u
Both supplied with:10X Buffer Bsp143I 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® Sau3Al (Bsp143I)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Bsp143I at 37°C.
Storage BufferBsp143I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Bsp143I more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – blocked (p.179).
NoteDpnI, Bsp143I and MboI all recognize the same sequence but have different methylation sensitivities (pp.175-181) and cleavage sites.
Bsp143I (Sau3AI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 0-20 0-20 50-100 20-50
100
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...T↓G T A C A...3’
3’...A C A T G↑T...5’
#ER0931 300 u#ER0932 1500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® Bsp1407I
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferBsp1407I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Bsp1407I more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded λ DNA in 16 hours.
Bsp1407I (BsrGI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 0-20 20-50 100 50-100
BspCNI Fermentas enzymes FastDigest® BspCNI (BseMII) and BseMII (BspCNI)
BspDI Fermentas enzymes FastDigest® ClaI (Bsu15I) and Bsu15I (ClaI)
BspEI Fermentas enzymes FastDigest® Kpn2I and Kpn2I (BspEI)
BspHI Fermentas enzymes FastDigest® BspHI (PagI) and PagI (BspHI)
BspLI (NlaIV) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 20-50 100 20-50
5’...G G N↓N C C...3’
3’...C C N↑N G G...5’
#ER1151 200 u#ER1152 1000 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® NlaIV (BspLI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferBspLI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BspLI, approxi-mately 90% of the DNA fragments can be ligated and more than 95% of these can be recut.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179).
NoteBspLI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
BspLU11I Fermentas enzyme PscI (PciI)
BspMI Fermentas enzymes FastDigest® BspMI (BveI) and BveI (BspMI)
BspMII Fermentas enzymes FastDigest® Kpn2I and Kpn2I (BspEI)
5’...G C T A G↓C...3’
3’...C↑G A T C G...5’
#ER2041 200 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BmtI (BspOI)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer O at 37°C.
Storage BufferBspOI is supplied in: 10 mM Tris-HCl (pH 8.2 at 25°C), 500 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.15% Triton X-100, 0.5 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BspOI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 20 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded lambda DNA in 16 hours.
BspOI (BmtI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 100 100 0-20 20-50
101
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...G G A T C(N)4↓...3’
3’...C C T A G(N)5↑...5’
#ER1321 100 u#ER1322 500 u
Both supplied with:10X Buffer Tango™ 1 ml
Concentration2 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 55°C.
Storage BufferBspPI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with BspPI, approxi-mately 70% of the DNA fragments can be ligated and more than 50% of these can be recut.
Methylation EffectsDam: completely overlaps – blocked (p.176).Dcm, CpG, EcoKI, EcoBI – no effect.
BspPI (AlwI) Activity in Five Buffer System, %Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 0-20 0-20 100 0-20
Note• Incubation at 37°C results in 30% activity.• BspPI is blocked by overlapping dam methy-
lation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
5’...C↓T T A A G...3’
3’...G A A T T↑C...5’
#ER0831 1000 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® AflII (BspTI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer O at 37°C.
Storage BufferBspTI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BspTI, more than 95% of the DNA fragments can be ligated and more than 95% of these can be recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
BspTI (AflII) B G O R Tango 2X Tango 0-20 0-20 100 20-50 0-20 50-100
Activity in Five Buffer System, %
BspQI Fermentas enzymes FastDigest® SapI (LguI) and LguI (SapI)
BspT107I Fermentas enzymes FastDigest® BanI (BshNI) and BshNI (BanI)
BsrI Fermentas enzymes FastDigest® BseNI and BseNI (BsrI)
BsrBI Fermentas enzymes FastDigest® BsrBI (MbiI) and MbiI (BsrBI)
BsrDI Fermentas enzymes FastDigest® BsrDI (BseMI) and BseMI (BsrDI)
BsrFI Fermentas enzymes FastDigest® BsrFI (Cfr10I) and Cfr10I (BsrFI)
BsrGI Fermentas enzymes FastDigest® Bsp1407I and Bsp1407I (BsrGI)
BsrSI Fermentas enzymes FastDigest® BseNI and BseNI (BsrI)
BssHII Fermentas enzyme FastDigest® BssHII (PteI) and PauI (BssHII)
BssKI Fermentas enzymes FastDigest® ScrFI (Bme1390I) and Bme1390I (ScrFI) (different cleavage position)
BssSI Fermentas enzyme BauI (BssSI)
BspT104I Fermentas enzymes FastDigest® Bsp119I and Bsp119I (BstBI)
Bst98I Fermentas enzymes FastDigest® AflII (BspTI) and BspTI (AflII)
102
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...G T A↓T A C...3’
3’...C A T↑A T G...5’
#ER0701 500 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BstZ17I (Bst1107I)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer O at 37°C.
Storage BufferBst1107I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Bst1107I, more than 90% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Bst1107I (BstZ17I) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 100 100 20-50 100
BstBI Fermentas enzymes FastDigest® Bsp119I and Bsp119I (BstBI)
BstEII Fermentas enzymes FastDigest® Eco91I and Eco91I (BstEII)
BstF5I Fermentas enzymes FastDigest® BseGI and BseGI (BtsCI); Fermentas enzyme FastDigest® FokI (different cleavage position)
BstNI Fermentas enzymes FastDigest® MvaI and MvaI (BstNI); EcoRII (different cleavage position and different sensitivity to methylation)
BstOI Fermentas enzymes FastDigest® MvaI and MvaI (BstNI); EcoRII (different cleavage position and different sensitivity to methylation)
BstPI Fermentas enzymes FastDigest® Eco91I and Eco91I (BstEII)
BstUI Fermentas enzymes FastDigest® Bsh1236I and Bsh1236I (BstUI)
BstXI Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 100 50-100 50-100 100
5’...C C A N N N N N↓N T G G...3’
3’...G G T N↑N N N N N A C C...5’
#ER1021 500 u#ER1022 2500 u
Both supplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BstXI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer O at 55°C.
Storage BufferBstXI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BstXI, approxi-mately 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, CpG, EcoKI, EcoBI – no effect.Dcm: may overlap – cleavage impaired (p.177).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Note• Incubation at 37°C results in 50% activity. • Greater than 15-fold overdigestion with
BstXI may result in star activity.• BstXI cleavage is impaired by overlapping dcm
methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
BstYI Fermentas enzymes FastDigest® PsuI and PsuI (BstYI)
BstZI Fermentas enzymes FastDigest® EagI (Eco52I) and Eco52I (EagI)
BstZ17I Fermentas enzymes FastDigest® BstZ17I (Bst1107I) and Bst1107I (BstZ17I)
103
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
Bsu36I Fermentas enzymes FastDigest® Bsu36I (Eco81I) and Eco81I (Bsu36I)
5’...G G↓C C...3’
3’...C C↑G G...5’
#ER0151 3000 uSupplied with:10X Buffer R 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® HaeIII (BsuRI)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer R at 37°C.
Storage BufferBsuRI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BsuRI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
BsuRI (HaeIII) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 50-100 100 50-100 100
Bsu15I (ClaI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 20-50 20-50 100 20-50
5’...A T↓C G A T...3’
3’...T A G C↑T A...5’
#ER0141 600 u#ER0145 1000 u
Both supplied with:10X Buffer Tango™ 1 ml
#ER0142 3000 uSupplied with:10X Buffer Tango™ 2x1 ml
Also available asFastDigest® ClaI (Bsu15I)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferBsu15I is supplied in: 10 mM Tris-HCl (pH 8.0 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Bsu15I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam: may overlap – blocked (p.176).Dcm, EcoKI – no effect.CpG: completely overlaps – blocked (p.178).EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NoteBsu15I is blocked by overlapping dam methy-lation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
BtrI Fermentas enzyme AjiI (BmgBI)
BtsCI Fermentas enzymes FastDigest® BseGI and BseGI (BtsCI); Fermentas enzyme FastDigest® FokI (different cleavage position)
104
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
NoteCaiI is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
5’...C A G N N N↓C T G...3’
3’...G T C↑N N N G A C...5’
#ER1391 500 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® AlwNI (CaiI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferCaiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with CaiI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, CpG, EcoKI – no effect.Dcm: may overlap – blocked (p.177).EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
CaiI (AlwNI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 20-50 50-100 100 50-100
CauII Fermentas enzymes FastDigest® NciI (BcnI) and BcnI (NciI)
CelII Fermentas enzymes FastDigest® BlpI (Bpu1102I) and Bpu1102I (BlpI)
CfoI Fermentas enzymes FastDigest® HinP1I (Hin6I) and Hin6I (HinP1I) (different cleavage position); FastDigest® HhaI and HhaI
5’...A C C T G C (N)4↓ ...3’
3’...T G G A C G (N)8↑ ...5’
#ER1741 250 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml50X oligonucleotide 2X25 µl
Also available asFastDigest® BspMI (BveI)
Concentration5 u/µl
Conditions for 100% Activity 1X Buffer O at 37°C.Oligonucleotide 0.5 µM.
Storage BufferBveI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 150 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with BveI, more than 90% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI: may overlap – effect not determined.
substrates with BveI is difficult to achieve.• BveI concentration is determined by the
maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.
• Low salt, high glycerol (>5%) concentra-tions, pH >8.0 or a large excess of enzyme may result in star activity.
Note• At least two copies of BveI recognition site
are required for efficient cleavage.• Inclusion of 0.5 µM oligonucleotide with the
BveI recognition sequence in the reaction mixture significantly improves cleavage of plasmid DNAs, especially of those with a single BveI site. Still, complete cleavage of some
• BveI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electro-phoresis.
BveI (BspMI) B G O R Tango 2X Tango 0-20 20-50 100 20-50 50-100 100
Activity in Five Buffer System, %
105
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
NoteCfrI is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
5’...Y↓G G C C R...3’
3’...R C C G G↑Y...5’
#ER0161 200 uSupplied with:10X Buffer Tango™ 1 ml
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferCfrI is supplied in: 10 mM Tris-HCl (pH 8.5 at 25°C), 500 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with CfrI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – blocked (p.177, 179).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
CfrI (EaeI) Activity in Five Buffer System, %
* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
B G O R Tango 2X Tango 50-100* 50-100 0-20 0-20 100 0-20
NoteTo achieve complete digestion of substrate with Cfr9I, the concentration of DNA should be no less than 50 µg/ml in the reaction buffer.
5’...C↓C C G G G...3’
3’...G G G C C↑C...5’
#ER0171 300 u#ER0172 1500 u
Both supplied with:10X Buffer Cfr9I 1 ml10X Buffer Tango™ 1 ml
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Cfr9I at 37°C.
Storage BufferCfr9I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 250 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Cfr9I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – cleavage impaired (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Cfr9I (XmaI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 0-20 20-50 0-20
Note• Greater than 5-fold overdigestion with
Cfr10I may result in star activity.• For cleavage with Cfr10I at least two copies
of its recognition sequence are required.
5’...R↓C C G G Y...3’
3’...Y G G C C↑R...5’
#ER0181 200 uSupplied with:10X Buffer Cfr10I 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BsrFI (Cfr10I)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Cfr10I at 37°C.
Storage BufferCfr10I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and Recutting After 5-fold overdigestion with Cfr10I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Cfr10I (BsrFI) Activity in Five Buffer System, %
* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
B G O R Tango 2X Tango 0-20 20-50 20-50 50-100* 20-50 50-100
106
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
ClaI Fermentas enzymes FastDigest® ClaI (Bsu15I) and Bsu15I (ClaI)
NoteCfr13I is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
5’...G↓G N C C...3’
3’...C C N G↑G...5’
#ER0191 1000 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® Sau96I (Cfr13I)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferCfr13I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Cfr13I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – blocked (p.177, 179).
Cfr13I (Sau96I) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 20-50 100 20-50
5’...C C G C↓G G...3’
3’...G G↑C G C C...5’
#ER0201 1200 u#ER0205 2000 u
Both supplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
#ER0202 5x1200 uSupplied with:10X Buffer B 2x1 ml10X Buffer Tango™ 1 ml
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer B at 37°C.
Storage BufferCfr42I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Cfr42I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 20 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded λ DNA in 16 hours.
Note• Certain sites in lambda DNA are difficult to
cleave with Cfr42I, the same as with its proto-type SacII. At least two copies of Cfr42I recogni-tion site are required for efficient cleavage.
• Cfr42I activity is affected by high salt con-centration. Trace amounts of sodium chloride remaining in the substrate DNA after completion of upstream applications may inhibit enzyme activity and result in impaired DNA cleavage.
Cfr42I (SacII) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 0-20 0-20 50-100 0-20
5’...C G↓G W C C G...3’
3’...G C C W G↑G C...5’
#ER0741 200 u#ER0742 1000 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® RsrII (CpoI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferCpoI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with CpoI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
CpoI (RsrII) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 50-100 20-50 100 50-100
107
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
CspI Fermentas enzymes FastDigest® RsrII (CpoI) and CpoI (RsrII)
5’...G↓T A C...3’
3’...C A T↑G...5’
#ER0211 1500 uSupplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® Csp6I
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer B at 37°C.
Storage BufferCsp6I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Csp6I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Csp6I (CviQI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 0-20 0-20 50-100 0-20
Csp45I Fermentas enzymes FastDigest® Bsp119I and Bsp119I (BstBI)
CviAII Fermentas enzymes FastDigest® NlaIII (Hin1II) and Hin1II (NlaIII) (different cleavage position)
CviQI Fermentas enzymes FastDigest® Csp6I and Csp6I (CviQI)
DdeI Fermentas enzymes FastDigest® DdeI (HpyF3I) and HpyF3I (DdeI)
Note• For cleavage with CseI at least two copies of its
recognition sequence are required.• Low salt, high glycerol (>5%) concen-
trations, pH >8.0 or a large excess of enzyme may result in star activity.
• CseI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
5’...G A C G C (N) 5↓...3’
3’...C T G C G (N)10↑...5’
#ER1901 100 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® HgaI (CseI)
Concentration5 u/µl
Conditions for 100% Activity 1X Buffer R at 37°C.
Storage BufferCseI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with CseI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: completely overlaps – blocked (p.178).EcoBI: may overlap – effect not determined.
CseI (HgaI) Activity in Five Buffer System, %
* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
B G O R Tango 2X Tango NR 50-100* 50-100 100 100* 50-100
108
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
DpnII Fermentas enzymes FastDigest® DpnI and DpnI (different cleavage position and different sensitivity to methylation); FastDigest® Sau3AI (Bsp143I) and Bsp143I (Sau3AI) (different sensitivity to methylation); FastDigest® MboI and MboI
5’...T T T↓A A A...3’
3’...A A A↑T T T...5’
#ER0221 1500 uSupplied with:10X Buffer Tango™ 1 ml
#ER0223 HC, 7500 uSupplied with:10X Buffer Tango™ 2x1 ml
Also available asFastDigest® DraI,
Concentration10 u/µl,50 u/µl, HC
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferDraI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.15% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with DraI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – blocked (p.181).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
DraI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 20-50 100 50-100
DraII Fermentas enzymes FastDigest® Eco0109I and EcoO109I
DraIII Fermentas enzymes FastDigest® DraIII (AdeI) and AdeI (DraIII)
DrdI Fermentas enzymes FastDigest® DrdI (AasI) and AasI (DrdI)
EaeI Fermentas enzyme CfrI (EaeI)
EagI Fermentas enzymes FastDigest® EagI (Eco52I) and Eco52I (EagI)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferDpnI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 400 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with DpnI, more than 70% of the DNA fragments can be ligated and more than 95% of these can be recut.
Methylation EffectsDam: does not cut dam– DNA (p.176).Dcm, EcoKI, CpG – no effect.EcoBI: may overlap – effect not determined.
Note• DpnI requires the presence of N6-methyladenine
within the recognition sequence to cleave DNA.• DNA purified from a dam+ strain will be a
substrate for DpnI.• DpnI will only cleave fully-adenomethylated
dam sites. Hemi-adenomethylated dam sites DpnI cleaves 60X more slowly.
• DpnI, Bsp143I and MboI all recognize the same sequence but have different methy-lation sensitivities (pp.175-181) and cleavage sites.
DpnI Activity in Five Buffer System, % B G O R Tango 2X Tango 100 100 50-100 50-100 100 50-100
5’...G m6A↓ T C...3’
3’...C T↑m6A G...5’
#ER1701 500 u#ER1705 1000 u#ER1702 2500 u
All supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® DpnI
109
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
EarI Fermentas enzymes FastDigest® EarI (Eam1104I) and Eam1104I (EarI)
NoteLow salt, high glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.
5’...G A C N N N↓N N G T C...3’
3’...C T G N N↑N N N C A G...5’
#ER0241 1000 uSupplied with:10X Buffer Eam1105I 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® Eam1105I
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Eam1105I at 37°C.
Storage BufferEam1105I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with Eam1105I, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Eam1105I (AhdI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 0-20 0-20 50-100 20-50
5’...G A G↓C T C...3’
3’...C T C↑G A G...5’
#ER0251 1500 uSupplied with:10X Buffer Ecl136II 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® Ecl136II
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Ecl136II at 37°C.
Storage BufferEcl136II is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Ecl136II, more than 90% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Ecl136II (EcoICRI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 0-20 0-20 50-100 0-20
NoteCertain sites in λ DNA are difficult to cleave with Eam1104I, the same as with its prototype Ksp632I.
5’...C T C T T C(N)1↓...3’
3’...G A G A A G(N)4↑...5’
#ER0231 300 u#ER0232 1500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® EarI (Eam1104I)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferEam1104I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Eam1104I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1µg of agarose-embed-ded λ DNA in 16 hours.
Eam1104I (EarI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 0-20 100 0-20
110
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...G R G C Y↓C...3’
3’...C↑Y C G R G...5’
#ER0281 1500 uSupplied with:10X Buffer Tango™ 1 ml
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferEco24I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Eco24I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Eco24I (BanII) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 20-50 100 0-20
EclHKI Fermentas enzymes FastDigest® Eam1105I and Eam1105I (AhdI)
EclXI Fermentas enzymes FastDigest® EagI (Eco52I) and Eco52I (EagI)
5’...G G T C T C(N)1↓...3’
3’...C C A G A G(N)5↑...5’
#ER0291 1000 uSupplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml
#ER0292 5000 uSupplied with:10X Buffer G 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® Eco31I
Concentration10 u/µl
Conditions for 100% Activity1X Buffer G at 37°C.
Storage BufferEco31I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Eco31I, more than 95% of DNA fragments can be ligated and recut.
Methylation EffectsDam, EcoKI – no effect.Dcm, CpG: may overlap – cleavage impaired (177, 179).EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Note• Eco31I cleavage is impaired by overlapping
dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
• Low salt, high glycerol (>5%) concentra-tions, pH >8.0 or a large excess of enzyme may result in star activity.
Eco31I (BsaI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 100 0-20 0-20 50-100 20-50
5’...G A T↓A T C...3’
3’...C T A↑T A G...5’
#ER0301 2000 uSupplied with:10X Buffer R 1ml10X Buffer Tango™ 1ml
#ER0305 4000 u#ER0302 5x2000 u#ER0303 HC, 10000 u
All supplied with:10X Buffer R 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® EcoRV (Eco32I)
Concentration10 u/µl50 u/µl, HC
Conditions for 100% Activity1X Buffer R at 37°C.
Storage BufferEco32I is supplied in: 25 mM Tris-HCl (pH 7.5 at 25°C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Eco32I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Eco32I (EcoRV) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 50-100 100 20-50 100
111
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
NoteEco47I is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
5’...G↓G W C C...3’
3’...C C W G↑G...5’
#ER0311 800 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
#ER0312 4000 uSupplied with:10X Buffer R 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® AvaII (Eco47I)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer R at 37°C.
Storage BufferEco47I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Eco47I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – blocked (p.177, 179).
Eco47I (AvaII) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 50-100 100 50-100 50-100
5’...A G C↓G C T...3’
3’...T C G↑C G A...5’
#ER0321 200 u#ER0322 1000 u
Both supplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® AfeI (Eco47III)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer O at 37°C.
Storage BufferEco47III is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Eco47III, more than 80% of DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: completely overlaps – blocked (p.178).EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Eco47III (AfeI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 100 50-100 100
5’...C↓G G C C G...3’
3’...G C C G G↑C...5’
#ER0331 500 u#ER0332 2500 u
Both supplied with:10X Buffer Eco52I 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® EagI (Eco52I)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Eco52I at 37°C.
Storage BufferEco52I is supplied in: 10 mM Tris-HCl (pH 8.2 at 25°C), 500 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Eco52I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Eco52I (EagI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 20-50 0-20 20-50
Eco53kI Fermentas enzymes FastDigest® Ecl136II and Ecl136II (EcoICRI); Fermentas enzymes FastDigest® SacI and SacI (different cleavage position)
112
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Ligation and RecleavageAfter 10-fold overdigestion with Eco57I, approximately 70% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
5’...C T G A A G(N)16↓...3’
3’...G A C T T C(N)14↑...5’
#ER0341 200 uSupplied with:10X Buffer G 1 ml50X SAM 0.1 ml10X Buffer Tango™ 1 ml
#ER0342 1000 uSupplied with:10X Buffer G 1 ml50X SAM 2x0.1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® AcuI (Eco57I)
Concentration5 u/µl
Conditions for 100% Activity 1X Buffer G at 37°C. SAM 0.01 mM.
Storage Buffer Eco57I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT and 50% (v/v) glycerol.
Note• Eco57I requires only Mg2+ for its activity, but is
stimulated by S-adenosylmethionine. 0.01 mM S-adenosylmethionine gives a 100-fold increase in Eco57I activity. Still, complete cleavage of some substrates with Eco57I is difficult to achieve.
• For cleavage with Eco57I at least two copies of its recognition sequence are required.
• Eco57I concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.
• Low salt, high glycerol (>5%) concentra-tions, pH >8.0 or a large excess of enzyme may result in star activity.
• Eco57I may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
Eco57I (AcuI) B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM
100 100 20-50 20-50 50-100 50-100
Activity in Five Buffer System, %
5’...C T G R A G(N)16↓...3’
3’...G A C Y T C(N)14↑...5’
#ER1671 50 uSupplied with:10X Buffer B 1 ml50X SAM 0.1 ml10X Buffer Tango™ 1 ml
Concentration2 u/µl
Conditions for 100% Activity 1X Buffer B at 37°C.SAM 0.01 mM.
Storage Buffer Eco57MI is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with Eco57MI, approximately 90% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.
Methylation EffectsDam, CpG, EcoKI – no effect.Dcm: may overlap – blocked (p.177).EcoBI: may overlap – effect not determined.
Note• Eco57MI requires only Mg2+ for its activity,
but is stimulated by S-adenosylmethionine. 0.01 mM S-adenosylmethionine gives more than a 100-fold increase in Eco57MI activity. Still, complete cleavage of some substrates with Eco57MI is difficult to achieve.
• Eco57MI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.
• Eco57MI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA
Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electro-phoresis.
• Eco57MI is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Eco57MI Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM
100 50-100 0-20 20-50 50-100 0-20
113
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
NoteGreater than 10-fold overdigestion with Eco72I may result in star activity.
5’...C A C↓G T G...3’
3’...G T G↑C A C...5’
#ER0361 2000 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® PmlI (Eco72I)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferEco72I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with Eco72I, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Eco72I (PmlI) Activity in Five Buffer System, % B G O R Tango 2X Tango NR NR 0-20 0-20 100 20-50
5’...C C↓T N A G G...3’
3’...G G A N T↑C C...5’
#ER0371 500 u#ER0372 2500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® Bsu36l (Eco81I)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferEco81I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with Eco81I, more than 80% of the DNA fragments can be ligated in a reaction mixture containing 20-40 u of T4 DNA Ligase/1 µg of fragments and 10% PEG. More than 95% of these can be recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Eco81I (Bsu36I) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 100 0-20 0-20 100 0-20
5’...C↓Y C G R G...3’
3’...G R G C Y↑C...5’
#ER0381 1000 uSupplied with: 10X Buffer Tango™ 1 ml
Also available asFastDigest® AvaI (Eco88I)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferEco88I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Eco88I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – cleavage impaired (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Eco88I (AvaI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 0-20 0-20 100 20-50
114
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...G↓G T N A C C...3’
3’...C C A N T G↑G...5’
#ER0391 1000 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
#ER0392 5000 uSupplied with:10X Buffer O 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® Eco91I
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer O at 37°C.
Storage BufferEco91I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Eco91I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG – no effect.EcoKI, EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Eco91I (BstEII) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 100 50-100 50-100 100
NoteGreater than 15-fold overdigestion with Eco105I may result in star activity.
5’...T A C↓G T A...3’
3’...A T G↑C A T...5’
#ER0401 600 uSupplied with:10X Buffer Tango™ 1 ml
#ER0402 3000 uSupplied with:10X Buffer Tango™ 2x1 ml
Also available asFastDigest® SnaBI (Eco105I)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferEco105I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with Eco105I, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Eco105I (SnaBI)* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
Activity in Five Buffer System, % B G O R Tango 2X Tango 100* 50-100 0-20 0-20 100 0-20
5’...C↓C W W G G...3’
3’...G G W W C↑C...5’
#ER0411 2500 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® StyI (Eco130I)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer O at 37°C.
Storage BufferEco130I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Eco130I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Eco130I (StyI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 50-100 50-100 100
115
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
NoteEco147I is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
5’...A G G↓C C T...3’
3’...T C C↑G G A...5’
#ER0421 1000 uSupplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
#ER0422 5000 uSupplied with:10X Buffer B 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® StuI (Eco147I)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer B at 37°C.
Storage BufferEco147I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Eco147I, more than 90% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, CpG, EcoKI – no effect.Dcm: may overlap – blocked (p.177).EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Eco147I (StuI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 20-50 20-50 50-100 0-20
NoteEcoO109I is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
5’...R G↓G N C C Y...3’
3’...Y C C N G↑G R...5’
#ER0261 2000 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® EcoO109I
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferEcoO109I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with EcoO109I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, CpG, EcoKI – no effect.Dcm: may overlap – blocked (p.177).EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme is required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
EcoO109I (DraII) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 20-50 20-50 100 100
EcoICRI Fermentas enzymes FastDigest® Ecl136II and Ecl136II (EcoICRI); FastDigest® SacI and SacI (different cleavage position)
EcoNI Fermentas enzymes FastDigest® EcoNI (XagI) and XagI (EcoNI)
EcoO65I Fermentas enzymes FastDigest® Eco91I and Eco91I (BstEII)
116
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
NoteLow salt concentration, large excess of the enzyme, pH >8.0, or the replacement of Mg2+ by Mn2+ may result in star activity.
5’...G↓A A T T C...3’
3’...C T T A A↑G...5’
#ER0271 5000 uSupplied with:10X Buffer EcoRI 2x1 ml10X Buffer Tango™ 1 ml
#ER0275 10000 uSupplied with:10X Buffer EcoRI 4x1 ml10X Buffer Tango™ 1 ml
#ER0272 5x5000 u#ER0273 HC, 25000 u
All supplied with:10X Buffer EcoRI 5x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® EcoRI
Concentration10 u/µl50 u/µl, HC
Conditions for 100% Activity1X Buffer EcoRI at 37°C.
Storage BufferEcoRI is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 300 mM NaCl, 1 mM EDTA, 1 mM DTT,0.2 mg/ml BSA, 0.15% Triton X-100 and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with EcoRI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
EcoRI Activity in Five Buffer System, %
* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
B G O R Tango 2X Tango 0-20 NR 100 100* NR 100
5’...↓C C W G G ...3’
3’... G G W C C↑...5’
#ER1921 200 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer O at 37°C.
Storage BufferEcoRII is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with EcoRII, more than 90% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, CpG, EcoKI, EcoBI – no effect.Dcm: completely overlaps – blocked (p.177).
Note• At least two copies of EcoRII recognition
site are required for efficient cleavage. For cleavage of DNA substrates with only one copy of recognition site MvaI (#ER0551), neoschizomer of EcoRII, is recommended.
• EcoRII may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
• EcoRII is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
EcoRII Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 100 50-100 20-50 50-100
EcoRV Fermentas enzymes FastDigest® EcoRV (Eco32I) and Eco32I (EcoRV)
EcoT14I Fermentas enzymes FastDigest® StyI (Eco130I) and Eco130I (StyI)
EcoT22I Fermentas enzymes FastDigest® NsiI (Mph1103I) and Mph1103I (NsiI)
117
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
EspI Fermentas enzymes FastDigest® BlpI (Bpu1102I) and Bpu1102I (BlpI)
Note• Unlike NarI, EheI completely digests λ and
pBR322 DNAs.• Low salt, high glycerol (>5%) concentra-
tions, pH >8.0 or a large excess of enzyme may result in star activity.
5’...G G C↓G C C...3’
3’...C C G↑C G G...5’
#ER0441 500 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® EheI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferEheI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with EheI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 20 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded λ DNA in 16 hours.
EheI (SfoI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 0-20 0-20 100 20-50
5’...C G T C T C(N)1↓...3’
3’...G C A G A G(N)5↑...5’
#ER0451 200 u#ER0452 1000 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® BsmBI (Esp3I)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.DTT (#R0861/2) 1 mM.
Storage BufferEsp3I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.5 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Esp3I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: completely overlaps – blocked (p.178).EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NoteThe enzyme requires DTT (#R0861/2). Freshly made DTT should be added to the reaction buffer.
Esp3I (BsmBI) Activity in Five Buffer System, % B+DTT G+DTT O+DTT R+DTT Tango+DTT 2X Tango+DTT
100 20-50 0-20 0-20 100 0-20
118
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
NoteLow salt, high glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.
5’...R T G C↓G C A Y...3’
3’...Y A C G↑C G T R...5’
#ER1661 100 u#ER1662 500 u
Both supplied with: 10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® FspAI
Concentration5 u/µl
Conditions for 100% Activity1X Buffer O at 37°C.
Storage BufferFspAI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with FspAI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not determined.
FspAI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 100 50-100 0-20 50-100
FatI Fermentas enzymes FastDigest® NlaIII (Hin1II) and Hin1II (different cleavage position)
FauI Fermentas enzyme SmuI (FauI)
FbaI Fermentas enzymes FastDigest® BclI and BclI
FinI Fermentas enzymes FastDigest® BsmFI (FaqI) and Faq (BsmFI)
FnuDII Fermentas enzymes FastDigest® Bsh1236I and Bsh1236I (BstUI)
Fnu4HI Fermentas enzymes FastDigest® Fnu4HI (SatI) and SatI (Fnu4HI)
FokI Fermentas enzymes FastDigest® FokI; FastDigest® BseGI and BseGI (BtsCI) (different cleavage position)
FspI Fermentas enzymes FastDigest® FspI (NsbI) and NsbI (FspI)
5’...G G G A C(N)10↓...3’
3’...C C C T G(N)14↑...5’
#ER1811 100 uSupplied with:10X Buffer Tango™ 1 ml50X SAM 0.1 ml
Also available asFastDigest® BsmFI (FaqI)
Concentration2 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.SAM 0.05 mM.
Storage BufferFaqI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with FaqI, more than 90% of the DNA fragments can be ligated, but none of these can be recut due to the methyla-tion of the recognition sequence by this enzyme.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – blocked (p.179).
Note• FaqI requires only Mg2+ for its activity, but
is stimulated by S-adenosylmethionine. 0.05 mM S-adenosylmethionine gives more than a 2-fold increase in FaqI activity. Still, complete cleavage of some substrates is difficult to achieve.
• For cleavage with FaqI at least two copies of its recognition sequence are required.
• FaqI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.
• FaqI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
FaqI (BsmFI) Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM
20-50 20-50 0-20 0-20 100 20-50
119
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...C↓T A G...3’
3’...G A T↑C...5’
#ER1761 500 u#ER1762 2500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® BfaI (FspBI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferFspBI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with FspBI, more than 80% of the DNA fragments can be ligated and more than 95% of these can be recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded λ DNA in 4 hours.
FspBI (BfaI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 0-20 0-20 100 0-20
HaeIII Fermentas enzymes FastDigest® HaeIII (BsuRI) and BsuRI (HaeIII)
HaeII Fermentas enzyme FastDigest® HaeII (BfoI)
HapII Fermentas enzymes FastDigest® HpaII and HpaII; FastDigest® MspI and MspI (HpaII) (different sensitivity to methylation)
HgaI Fermentas enzymes FastDigest® HgaI (CseI) and CseI (HgaI)
HgiAI Fermentas enzymes FastDigest® Alw21I and Alw21I (BsiHKAI)
HgiCI Fermentas enzymes FastDigest® BanI (BshNI) and BshNI (BanI)
HgiJII Fermentas enzyme Eco24I
5’...C T G G A G(N)16↓...3’
3’...G A C C T C(N)14↑...5’
#ER0461 100 u#ER0462 500 u
Both supplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BpmI (GsuI)
Concentration5 u/µl
Conditions for 100% Activity1X Buffer B at 30°C.
Storage BufferGsuI is supplied in: 10 mM potassium phosphate (pH 7.5 at 25°C), 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with GsuI, approxi-mately 90% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, CpG, EcoKI, EcoBI – no effect.Dcm: may overlap – blocked (p.177).
Note• Incubation at 37°C results in 70% activity.• GsuI requires only Mg2+ for its activity, but
is stimulated by S-adenosylmethionine. 0.01 mM S-adenosylmethionine gives a 2-fold increase in activity.
• GsuI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
• GsuI is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
GsuI (BpmI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 20-50 20-50 100 50-100
GsaI Fermentas enzyme FastDigest® PspFI
120
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...G C G↓C...3’
3’...C↑G C G...5’
#ER1851 2000 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® HhaI
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferHhaI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with HhaI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
HhaI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 20-50 100 20-50
NoteHin1I cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
5’...G R↓C G Y C...3’
3’...C Y G C↑R G...5’
#ER0471 300 uSupplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BsaHI (Hin1I)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer G at 37°C.
Storage BufferHin1I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Hin1I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, EcoKI – no effect.Dcm: may overlap – cleavage impaired (p.177).CpG: completely overlaps – blocked (p.178).EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Hin1I (BsaHI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 20-50 20-50 20-50 20-50
NoteMore stable when stored at -70°C. At -20°C the half-life of Hin1II is 6 months.
5’... C A T G↓...3’
3’...↑G T A C ...5’
#ER1831 300 uSupplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® NlaIII (Hin1II)
Concentration5 u/µl
Conditions for 100% Activity1X Buffer G at 37°C.
Storage BufferHin1II is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 500 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.15% Triton X-100, 0.5 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Hin1II, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Hin1II (NlaIII) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 100 20-50 50-100 50-100 50-100
121
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
HinP1I Fermentas enzymes FastDigest® HhaI and HhaI (different cleavage position); FastDigest® HinP1I (Hin6I) and Hin6I (HinP1I)
5’...↓8 (N)GAY(N)5VTC(N)13-14↓...3’
3’...↑13-14(N)CTR(N)5BAG(N)8 ↑...5’
#ER1601 50 uSupplied with:10X Buffer Tango™ 1 ml50X SAM 0.1 ml
Concentration2 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.SAM 0.05 mM.
Storage BufferHin4I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with Hin4I, more than 95% of the DNA fragments can be ligated and only 50% of these can be recut due to the methylation of the recognition sequence by restriction enzyme.
Methylation EffectsDam: may overlap – blocked (p.176).Dcm, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – effect not determined.
Note• Hin4I requires only Mg2+ for its activity,
but is stimulated by S-adenosylmethionine. 0.05 mM S-adenosylmethionine gives more than a 10-fold increase in Hin4I activity. Still, complete cleavage of some substrates with Hin4I is difficult to achieve.
• Hin4I concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.
• Hin4I produces double-strand cuts on both sides from the interrupted recognition site.
Its unique feature is a degenerate cleavage point on the 3’ side of the recognition sequence (13 or 14 nt away).
• Hin4I is blocked by overlapping dam methy-lation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Hin4I Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM
20-50 20-50 0-20 0-20 100 0-20
5’...G↓C G C...3’
3’...C G C↑G...5’
#ER0481 2000 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® HinP1I (Hin6I)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferHin6I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Hin6I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Hin6I (HinP1I) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 20-50 100 50-100
HindII Fermentas enzymes FastDigest® HincII and HincII (HindII)
5’...G T Y↓R A C...3’
3’...C A R↑Y T G...5’
#ER0491 500 u#ER0492 2500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® HincII
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferHincII is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with HincII, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI: may overlap – blocked (p.181).
HincII (HindII) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 50-100 100 50-100
122
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...A↓A G C T T...3’
3’...T T C G A↑A...5’
#ER0501 5000 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
#ER0505 10000 uSupplied with:10X Buffer R 4x1 ml10X Buffer Tango™ 1 ml
#ER0502 5x5000 u#ER0503 HC, 25000 u
Both supplied with:10X Buffer R 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® HindIII
Concentration10 u/µl50 u/µl, HC
Conditions for 100% Activity 1X Buffer R at 37°C.
Storage BufferHindIII is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 250 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with HindIII, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – cleavage impaired (p.181).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
HindIII Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 0-20 100 50-100 50-100
5’...G↓A N T C...3’
3’...C T N A↑G...5’
#ER0801 2000 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
#ER0802 5x2000 u#ER0803 HC, 10000 u
Both supplied with:10X Buffer R 2x1 ml10X Buffer Tango™ 1 ml
FastDigest® HinfI
Concentration10 u/µl50 u/µl, HC
Conditions for 100% Activity 1X Buffer R at 37°C.
Storage BufferHinfI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with HinfI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – blocked (p.181).
HinfI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 50-100 100 50-100 50-100
HpaI Fermentas enzymes FastDigest® HpaI (KspAI) and KspAI (HpaI)
5’...C↓C G G...3’
3’...G G C↑C...5’
#ER0511 1000 uSupplied with:10X Buffer Tango™ 1 ml
#ER0512 5000 uSupplied with:10X Buffer Tango™ 2x1 ml
Also available asFastDigest® HpaII
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferHpaII is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with HpaII, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
HpaII Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 20-50 100 20-50
123
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
Note• HphI is blocked by overlapping dam methy-
lation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
• High glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.
5’...G G T G A(N)8↓...3’
3’...C C A C T(N)7↑...5’
#ER1101 300 u#ER1102 1500 u
Both supplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer B at 37°C.
Storage BufferHphI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with HphI, more than 70% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, EcoBI: may overlap – blocked (p.176, 181).Dcm, CpG, EcoKI – no effect.
HphI Activity in Five Buffer System, % B G O R Tango 2X Tango 100 0-20 0-20 0-20 20-50 0-20
5’...G T N↓N A C...3’
3’...C A N↑N T G...5’
#ER1571 200 u#ER1572 1000 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® Hpy8I
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferHpy8I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Hpy8I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI: may overlap – effect not determined.
Hpy8I (MjaIV) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 20-50 100 50-100
HpyCH4III Fermentas enzymes FastDigest® TaaI and TaaI (HpyCH4III)
HpyCH4IV Fermentas enzymes FastDigest® TaiI and TaiI (MaeII) (different cleavage position)
5’...C↓T N A G...3’
3’...G A N T↑C...5’
#ER1881 500 u#ER1882 2500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® DdeI (HpyF3I)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferHpyF3I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with HpyF3I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
HpyF3I (DdeI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 20-50 20-50 100 50-100
124
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...G C N N N N N↓N N G C...3’
3’...C G N N↑N N N N N C G...5’
#ER1731 300 u#ER1732 1500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® HpyF10VI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferHpyF10VI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with HpyF10VI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
HpyF10VI (MwoI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 0-20 100 50-100
Hsp92I Fermentas enzymes FastDigest® BsaHI (Hin1I) and Hin1I (BsaHI)
Hsp92II Fermentas enzymes FastDigest® NlaIII (Hin1II) and Hin1II (NlaIII)
ItaI Fermentas enzymes FastDigest® Fnu4HI (SatI) and SatI (Fnu4HI)
KasI Fermentas enzymes FastDigest® EheI and EheI (SfoI) (different cleavage position) Fermentas enzyme SspDI (KasI)
NoteHigh glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.
5’...G G T A C↓C...3’
3’...C↑C A T G G...5’
#ER0521 4000 uSupplied with:10X Buffer KpnI 2x1 ml10X Buffer Tango™ 1 ml
#ER0522 5x4000 u#ER0523 HC, 20000 u
Both supplied with:10X Buffer KpnI 4x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® KpnI
Concentration10 u/µl50 u/µl, HC
Conditions for 100% Activity1X Buffer KpnI at 37°C.
Storage BufferKpnI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with KpnI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
KpnI Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 0-20 0-20 0-20 20-50 0-20
NoteIncubation at 37°C results in 50% activity.
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 55°C.
Storage BufferKpn2I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Kpn2I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
5’...T↓C C G G A...3’
3’...A G G C C↑T...5’
#ER0531 500 u#ER0532 2500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® Kpn2I
Kpn2I (BspEI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 20-50 100 50-100
125
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
Note• Greater than 10-fold overdigestion with
Lsp1109I may result in star activity.• Lsp1109I may remain associated with the
cleaved DNA. This may cause DNA band shift-ing during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
5’...G C A G C(N) 8↓...3’
3’...C G T C G(N)12↑...5’
#ER2071 200 uSupplied with:10X Buffer Lsp1109I 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BbvI (Lsp1109I)
Concentration5 u/µl
Conditions for 100% Activity1X Buffer Lsp1109I at 37°C.
Storage BufferLsp1109I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with Lsp1109I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
KspI Fermentas enzyme Cfr42I (SacII)
Ksp632I Fermentas enzymes FastDigest® EarI (Eam1104I) and Eam1104I (EarI)
NoteHigh glycerol (>5%) concentration, pH >8.0 or a large excess of enzyme may result in star activity.
5’...G T T↓A A C...3’
3’...C A A↑T T G...5’
#ER1031 500 u#ER1032 2500 u
Both supplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® HpaI (KspAI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer B at 37°C.
Storage BufferKspAI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with KspAI, more than 90% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI: may overlap – blocked (p.181).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
KspAI (HpaI) Activity in Five Buffer System, %
* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
B G O R Tango 2X Tango 100 50-100* 20-50 20-50 100* 50-100
NoteLow salt, high glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.
5’...G C T C T T C(N)1↓...3’
3’...C G A G A A G(N)4↑...5’
#ER1931 100 u#ER1932 500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® SapI (LguI)
Concentration5 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferLguI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with LguI, more than 90% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
LguI (SapI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 20-50 20-50 100 20-50
Lsp1109I (BbvI) Activity in Five Buffer System, %
* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
B G O R Tango 2X Tango 0-20 20-50* 50-100* 100* 20-50* 20-50*
126
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
MaeI Fermentas enzymes FastDigest® BfaI (FspBI) and FspBI (BfaI)
MaeII Fermentas enzymes FastDigest® TaiI and TaiI (MaeII) (different cleavage position)
MamI Fermentas enzymes FastDigest® BsaBI (BseJI) and BseJI (BsaBI)
Note• At least two copies of LweI recognition site
are required for efficient cleavage.• LweI may remain associated with the cleaved
DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
5’...G C A T C(N)5↓...3’
3’...C G T A G(N)9↑...5’
#ER1621 100 u#ER1622 500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® SfaNI (BmsI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferLweI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with LweI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI– no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – effect not determined.
LweI (SfaNI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 20-50 100 20-50
5’...C G↓C G C G C G...3’
3’...G C G C G C↑G C...5’
#ER2081 100 uSupplied with:10X Buffer Tango™ 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® MauBI
Concentration5 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferMauBI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with MauBI, more than 90% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
MauBI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 0-20 100 0-20
NoteLow salt, high glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.
5’...C C G↓C T C...3’
3’...G G C↑G A G...5’
#ER1271 1000 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® BsrBI (MbiI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferMbiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with MbiI, approxi-mately 80% of the DNA fragments can be ligated. No more than 50% of these can be recut due to the asymmetric recognition sequence of MbiI.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: completely overlaps – cleavage impaired (p.178).EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
MbiI (BsrBI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 20-50 20-50 100 20-50
127
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
Note• MboI is blocked by overlapping dam methy-
lation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
• MboI, Bsp143I and DpnI all recognize the same sequence but have different methy-lation sensitivities (pp.175-181) and cleavage sites.
5’...↓G A T C ...3’
3’... C T A G↑...5’
#ER0811 300 u#ER0812 1500 u
Both supplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® MboI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer R at 37°C.
Storage BufferMboI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with MboI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam: completely overlaps – blocked (p.176).Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – blocked (p.181).
MboI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 50-100 100 50-100 100
5’...G A A G A(N)8↓...3’
3’...C T T C T(N)7↑...5’
#ER0821 300 u#ER0822 1500 u
Both supplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® MboII
Concentration5 u/µl
Conditions for 100% Activity 1X Buffer B at 37°C.
Storage BufferMboII is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 5-fold overdigestion with MboII, more than 80% of the DNA fragments can be ligated and more than 80% of these can be recut.
Methylation EffectsDam: may overlap – blocked (p.176).Dcm, CpG, EcoKI, EcoBI – no effect.
Note• MboII is blocked by overlapping dam methy-
lation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
• Greater than 15-fold overdigestion with MboII may result in star activity.
• MboII produces DNA fragments that have a single-base 3’-extension which are more difficult to ligate than blunt-ended fragments.
• MboII may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample prepara-tion or heat the digested DNA in the presence of SDS prior to electrophoresis.
MboII Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 20-50 0-20 50-100 20-50
McrI Fermentas enzymes FastDigest® BsiEI (Bsh1285I) and Bsh1285I (BsiEI)
MfeI Fermentas enzymes FastDigest® MfeI (MunI) and MunI (MfeI)
MflI Fermentas enzymes FastDigest® PsuI and PsuI (BstYI)
MjaIV Fermentas enzymes FastDigest® Hpy8I and Hpy8I (MjaIV)
128
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
NoteMlsI is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
5’...T G G↓C C A...3’
3’...A C C↑G G T...5’
#ER1211 200 u#ER1212 1000 u
Both supplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® MscI (MlsI)
Concentration5 u/µ
Conditions for 100% Activity1X Buffer R at 37°C.
Storage BufferMlsI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with MlsI, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.
Methylation EffectsDam, CpG, EcoKI, EcoBI – no effect.Dcm: may overlap – blocked (p.177).
Digestion of Agarose-embedded DNAMinimum 20 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded λ DNA in 16 hours.
MlsI (MscI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 0-20 100 20-50 50-100
5’...A↓C G C G T...3’
3’...T G C G C↑A...5’
#ER0561 1000 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
#ER0562 5000 uSupplied with:10X Buffer R 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® MluI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer R at 37°C.
Storage BufferMluI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with MluI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
MluI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 50-100 100 20-50 50-100
MluNI Fermentas enzymes FastDigest® MscI (MlsI) and MlsI (MscI)
MlyI Fermentas enzymes FastDigest® MlyI (SchI) and SchI (MlyI)
Note• MnlI produces DNA fragments that have a
single-base 3’-extension which are more difficult to ligate than blunt-ended fragments.
• MnlI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
5’...C C T C(N)7↓...3’
3’...G G A G(N)6↑...5’
#ER1071 300 u#ER1072 1500 u
Both supplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® MnlI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer G at 37°C.
Storage BufferMnlI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with MnlI, approxi-mately 90% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – blocked (p.181).
MnlI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 100 20-50 20-50 20-50 20-50
129
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...A T G C A↓T...3’
3’...T↑A C G T A...5’
#ER0731 1000 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
#ER0732 5000 uSupplied with:10X Buffer R 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® NsiI (Mph1103I)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer R at 37°C.
Storage BufferMph1103I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 200 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Mph1103I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Mph1103I (NsiI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 20-50 100 50-100 50-100
5’...C G↓C C G G C G...3’
3’...G C G G C C↑G C...5’
#ER2021 300 uSupplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® MreI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer G at 37°C.
Storage BufferMreI is supplied in: 10 mM potassium phosphate (pH 7.0 at 25°C), 200 mM KCl, 0.1 mM EDTA, 0.01% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with MreI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
MreI (Sse232I) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 0-20 0-20 50-100 0-20
MroI Fermentas enzymes FastDigest® Kpn2I and Kpn2I (BspEI)
MscI Fermentas enzymes FastDigest® MscI (MlsI) and MlsI (MscI)
MseI Fermentas enzymes FastDigest® Tru1I and Tru1I (MseI); Fermentas enzyme FastDigest® MseI (SaqAI)
MslI Fermentas enzymes FastDigest® MslI (RseI) and RseI (MslI)
NoteMspI is an isoschizomer of HpaII. When the external C in the sequence CCGG is methy-lated, MspI and HpaII cannot cleave. However, unlike HpaII, MspI can cleave the sequence when the internal C residue is methylated.
5’...C↓C G G...3’
3’...G G C↑C...5’
#ER0541 3000 uSupplied with:10X Buffer Tango™ 2X1 ml
#ER0542 5x3000 uSupplied with:10X Buffer Tango™ 2x1 ml
Also available asFastDigest® MspI
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferMspI is supplied in: 10 mM potassium phosphate (pH 7.5 at 25°C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with MspI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
MspI (HpaII) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 0-20 100 50-100
130
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...C↓A A T T G...3’
3’...G T T A A↑C...5’
#ER0751 300 u#ER0752 1500 u
Both supplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® MfeI (MunI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer G at 37°C.
Storage BufferMunI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with MunI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
MunI (MfeI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 100 0-20 0-20 100 0-20
5’...G T T T↓A A A C...3’
3’...C A A A↑T T T G...5’
#ER1341 250 u#ER1342 1250 u
Both supplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® MssI
Concentration5 u/µl
Conditions for 100% Activity1X Buffer B at 37°C.
Storage BufferMssI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with MssI, more than 90% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – blocked (p.181).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
MssI (PmeI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 0-20 0-20 0-20 20-50 0-20
MstI Fermentas enzymes FastDigest® FspI (NsbI) and NsbI (FspI)
Note• Low salt, high glycerol (>5%) concentrations
or a large excess of enzyme may result in star activity.
• Unlike its neoschizomer EcoRII, MvaI does not require multiple copies of recognition site for efficient cleavage.
5’...C C↓W G G...3’
3’...G G W↑C C...5’
#ER0551 2000 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® MvaI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer R at 37°C.
Storage BufferMvaI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 400 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with MvaI, more than 90% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
MvaI (BstNI) Activity in Five Buffer System, %
* Star activity appears at a greater than 5-fold overdigestion (5u x 1h).
B G O R Tango 2X Tango 20-50 20-50 50-100 100 20-50* 100
131
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...G A A T G C N↓...3’
3’...C T T A C↑G N ...5’
#ER0961 200 u#ER0962 1000 u
Both supplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® Mva1269I
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer R at 37°C.
Storage BufferMva1269I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Mva1269I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Mva1269I (BsmI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 50-100 100 0-20 50-100
MvnI Fermentas enzymes FastDigest® Bsh1236I and Bsh1236I (BstUI)
MwoI Fermentas enzymes FastDigest® HpyF10VI and HpyF10VI (MwoI)
NaeI Fermentas enzymes FastDigest® NaeI (PdiI) and PdiI (NaeI)
NarI Fermentas enzymes FastDigest® EheI and EheI (SfoI) (different cleavage position); Fermentas enzyme SspDI (KasI) (different cleavage position)
NciI Fermentas enzymes FastDigest® NciI (BcnI) and BcnI (NciI)
NoteLow salt, high glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.
5’...C↓C A T G G...3’
3’...G G T A C↑C...5’
#ER0571 500 u#ER0575 1000 u#ER0572 2500 u
All supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® NcoI
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferNcoI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with NcoI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NcoI Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 20-50 50-100 100 100
132
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...C A↓T A T G...3’
3’...G T A T↑A C...5’
#ER0581 500 u#ER0582 2500 u
Both supplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
#ER0585 4000 uBoth supplied with:10X Buffer O 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® NdeI,
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer O at 37°C.
Storage BufferNdeI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with NdeI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NdeI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 100 50-100 0-20 50-100
NdeII Fermentas enzymes FastDigest® Sau3AI (Bsp143I) and Bsp143I (Sau3AI) (different sensitivity to methylation); FastDigest® DpnI and DpnI (different cleavage position and different sensitivity to methylation); FastDigest® MboI and MboI
NgoMIV Fermentas enzymes FastDigest® NaeI (PdiI) and PdiI (NaeI) (different cleavage position)
Note• Low salt, high glycerol (>5%) concentra-
tions, pH >8.0 or a large excess of enzyme may result in star activity.
• NheI is inhibited by salt concentrations above 100 mM.
• Supercoiled plasmids may require up to 10-fold more NheI for complete digestion than linear DNAs (e.g. 10 units are required to cleave 1 µg of pBR322 DNA).
5’...G↓C T A G C...3’
3’...C G A T C↑G...5’
#ER0971 500 u#ER0975 1000 u#ER0972 2500 u
All supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® NheI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferNheI is supplied in: 10 mM Tris-HCl (pH 8.0 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and Recleavage After 50-fold overdigestion with NheI, more than 95% of DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NheI Activity in Five Buffer System, % B G O R Tango 2X Tango 100 20-50 0-20 0-20 100 0-20
NlaIII Fermentas enzymes FastDigest® NlaIII (Hin1II) and Hin1II (NlaIII)
NlaIV Fermentas enzymes FastDigest® NlaIV (BspLI) and BspLI (NlaIV)
133
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...↓G T S A C ...3’
3’... C A S T G↑...5’
#ER1511 200 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® NmuCI
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer R at 37°C.
Storage BufferNmuCI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with NmuCI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – effect not determined.
NmuCI (Tsp45I) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 50-100 100 20-50 50-100
5’...G C↓G G C C G C...3’
3’...C G C C G G↑C G...5’
#ER0591 300 u#ER0595 500 u#ER0592 1500 u#ER0593 HC, 1500 u
All supplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® NotI
Concentration10 u/µl50 u/µl, HC
Conditions for 100% Activity1X Buffer O at 37°C.
Storage BufferNotI is supplied in: 20 mM Tris-HCl (pH 7.8 at 25°C), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.02% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with NotI, more than 95% of DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded Adenovirus-2 DNA in 16 hours.
NoteSupercoiled plasmids may require up to 5-fold more NotI for complete digestion than linear DNAs.
NotI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 100 20-50 0-20 20-50
NruI Fermentas enzymes FastDigest® NruI (RruI) and Bsp68I (NruI)
5’...T G C↓G C A...3’
3’...A C G↑C G T...5’
#ER1221 400 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® FspI (NsbI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferNsbI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with NsbI, more than 80% of the DNA fragments can be ligated and more tha 95% of these can be recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NsbI (FspI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 0-20 20-50 100 20-50
NsiI Fermentas enzymes FastDigest® Nsil (Mph1103I) and Mph1103I (NsiI)
NspI Fermentas enzymes FastDigest® NspI (XceI) and XceI (NspI)
NspV Fermentas enzymes FastDigest® Bsp119I and Bsp119I (BstBI)
134
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...C A C N N↓N N G T G...3’
3’...G T G N N↑N N C A C...5’
#ER1631 200 u#ER1632 1000 u
Both supplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® AleI (OliI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer R at 37°C.
Storage BufferOliI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with OliI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI: may overlap– blocked (p.181).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
OliI (AleI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 100 0-20 50-100
5’...G C A T G↓C...3’
3’...C↑G T A C G...5’
#ER0601 500 u#ER0602 2500 u
Both supplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® SphI (PaeI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer B at 37°C.
Storage BufferPaeI is supplied in: 10 mM potassium-phosphate (pH 7.0 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.15% Triton X-100, 0.5 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with PaeI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – blocked (p.181).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
PaeI (SphI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 0-20 0-20 50-100 0-20
PaeR7I Fermentas enzymes FastDigest® XhoI and XhoI
PacI Activity in Five Buffer System, %
5’...T T A A T↓T A A...3’
3’...A A T↑T A A T T...5’
#ER2201 250 u#ER2202 1250 u
Supplied with:10X Buffer PacI 1 ml
Also available asFastDigest® PacI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer PacI at 37°C.
Storage BufferPacI is supplied in: 10 mM potassium phosphate (pH 7.5 at 25°C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with PacI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded pJET1 DNA with inserted PacI recognition sequence in 16 hours.
B G O R Tango 2X Tango 20-50 20-50 0-20 0-20 0-20 0-20
135
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...C C↓C W G G G...3’
3’...G G G W C↑C C...5’
#ER1861 200 uSupplied with:10X Buffer PasI 1 ml
Concentration10 u/µl
Conditions for 100% Activity1X Buffer PasI at 55°C.
Storage BufferPasI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with PasI, more than 90% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded λ DNA in 16 hours.
Note• Incubation at 37°C results in 30% activity.• Greater than 10-fold overdigestion with PasI
results in star activity.
PasI Activity in Five Buffer System, % B G O R Tango 2X Tango NR NR NR NR NR NR
5’...T↓C A T G A...3’
3’...A G T A C↑T...5’
#ER1281 400 u#ER1282 2000 u
Both supplied with:10X Buffer O 1 ml
Also available asFastDigest® BspHI (PagI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer O at 37°C.
Storage BufferPagI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with PagI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, EcoBI: may overlap – cleavage impaired (p.176, 181).Dcm, CpG, EcoKI – no effect.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Note• Low salt, high glycerol (>5%) concentra-
tions, pH >8.0 or a large excess of enzyme may result in star activity.
• PagI cleavage is impaired by overlapping dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
PagI (BspHI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 100 NR NR NR
5’...G↓C G C G C...3’
3’...C G C G C↑G...5’
#ER1091 200 u#ER1092 1000 u
Both supplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® BssHII (PteI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer R at 37°C.
Storage BufferPauI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with PauI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded λ DNA in 16 hours.
NoteLow salt, high glycerol (>5%) concentrations or a large excess of enzyme may result in star activity.
PauI (BssHII) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 100 100 0-20 100
136
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
PciI Fermentas enzyme PscI (PciI)
5’...G A A N N↓N N T T C...3’
3’...C T T N N↑N N A A G...5’
#ER1531 500 u#ER1532 2500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® PdmI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferPdmI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl,1 mM DTT, 5 mM MgCl2, 0.2 mg/ml BSAand 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with PdmI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded λ DNA in 16 hours.
PdmI (XmnI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 0-20 0-20 100 0-20
5’...G C C↓G G C...3’
3’...C G G↑C C G...5’
#ER1521 200 u#ER1522 1000 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® NaeI (PdiI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferPdiI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 500 mM KCl,1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA, 0.15% Triton X-100 and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with PdiI, more than 90% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 20 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded pBR322 DNA in 16 hours.
Note• For cleavage with PdiI at least two copies of its
recognition sequence are required.• Certain sites in pBR322 are difficult to cleave
with PdiI, the same as with its prototype NaeI.
PdiI (NaeI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 0-20 0-20 100 50-100
5’...G↓A W T C...3’
3’...C T W A↑G...5’
#ER1781 500 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® TfiI (PfeI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer O at 37°C.
Storage BufferPfeI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 250 mM KCl,1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSAand 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with PfeI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – blocked (p.179).EcoBI: may overlap – effect not determined.
PfeI (TfiI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 50-100 20-50 50-100
137
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...T↓C C N G G A...3’
3’...A G G N C C↑T...5’
#ER1751 200 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® PfoI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferPfoI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with PfoI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam: may overlap – cleavage impaired (p.176).Dcm, CpG: may overlap – blocked (p.177, 179).EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded λ DNA in 16 hours.
NotePfoI cleavage is impaired by overlapping dam methylation and blocked by overlapping dcm methylation. To avoid dam or dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
PfoI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 50-100 0-20 100 50-100
PflFI Fermentas enzymes FastDigest® PsyI and PsyI (Tth111I)
PflMI Fermentas enzymes FastDigest® PflMI (Van91I) and Van91I (PflMI)
5’...C↓G T A C G...3’
3’...G C A T G↑C...5’
#ER0851 300 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® BsiWI (Pfl23II)
Concentration3 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferPfl23II is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with Pfl23II, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Pfl23II (BsiWI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 20-50 20-50 100 0-20
PhoI Fermentas enzymes FastDigest® HaeIII (BsuRI) and BsuRI (HaeIII)
PinAI Fermentas enzymes FastDigest® AgeI (BshTI) and BshTI (AgeI)
PleI Fermentas enzymes FastDigest® MlyI (SchI) and SchI (MlyI) (different cleavage position)
PmaCI Fermentas enzymes FastDigest® PmlI (Eco72I) and Eco72I (PmlI)
PmeI Fermentas enzymes FastDigest® MssI and MssI (PmeI)
PmlI Fermentas enzymes FastDigest® PmlI (Eco72I) and Eco72I (PmlI)
138
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...↓ 7(N)G A A C(N)5C T C(N)13↓...3’
3’...↑12(N)C T T G(N)5G A G(N) 8 ↑...5’
#ER1541 50 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Concentration2 u/µl
Conditions for 100% Activity1X Buffer R at 30°C.
Storage BufferPpiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with PpiI, more than 90% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
Note• Incubation at 37°C results in 60% activity.• PpiI cleaves certain DNA sequences at
random 7 or 8 nt away on the top strand of the recognition sequence:
5’...↓7-8(N) G A A C (N)5 C T C (N)13↓...3’ 3’...↑ 12(N) C T T G (N)5 G A G (N)8 ↑...5’• The presence of SAM in a reaction mixture
results in incomplete cleavage with PpiI.• Greater than 10-fold overdigestion with PpiI
may result in star activity.
PpiI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 100 50-100 50-100
PpuMI Fermentas enzymes FastDigest® PpuMI (Psp5II) and Psp5II (PpuMI)
5’...A↓C A T G T...3’
3’...T G T A C↑A...5’
#ER1871 200 u#ER1872 1000 u
Both supplied with:10X Buffer Tango™ 1 ml
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferPscI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with PscI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG – no effect.EcoKI, EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NotePscI, like its isoschizomers BspLU11I and PciI, is inhibited by nonionic detergents Triton X-100 (>0.002%) and Nonidet P40 (>0.001%).
PscI (PciI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 0-20 0-20 100 0-20
5’...Y A C↓G T R...3’
3’...R T G↑C A Y...5’
#ER1971 500 uSupplied with:10X Buffer Ppu21I 1 ml
Also available asFastDigest® BsaAI (Ppu21I)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Ppu21I at 30°C.
Storage BufferPpu21I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Ppu21I, more than 90% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Note• Incubation at 37°C results in less than 30%
activity.• Low salt, high glycerol (>5%) concentra-
tions, pH >8.0 or a large excess of enzyme may result in star activity.
* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
Ppu21I (BsaAI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100* 100* 20-50 NR NR NR
139
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...R G↓G W C C Y...3’
3’...Y C C W G↑G R...5’
#ER0761 500 uSupplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® PpuMI (Psp5II)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer G at 37°C.
Storage BufferPsp5II is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.15% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Psp5II, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, CpG, EcoKI – no effect.Dcm: may overlap – blocked (p.177).EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NotePsp5II is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Psp5II (PpuMI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 100 20-50 20-50 50-100 100
PshAI Fermentas enzymes FastDigest® PshAI (BoxI) and BoxI (PshAI)
PshBI Fermentas enzymes FastDigest® AseI (VspI) and VspI (AseI)
PsiI Fermentas enzymes FastDigest® PsiI (AanI) and AanI (PsiI)
5’...A A↓C G T T...3’
3’...T T G C↑A A...5’
#ER0941 300 u#ER0942 1500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® AclI (Psp1406I)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferPsp1406I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Psp1406I, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded λ DNA in 16 hours.
Psp1406I (AclI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 0-20 20-50 100 0-20
PspGI Fermentas enzymes EcoRII, FastDigest® MvaI and MvaI (BstNI) (different cleavage position and different sensitivity to methylation)
PspOMI Fermentas enzymes FastDigest® ApaI and ApaI (different cleavage position); FastDigest® Bsp120I and Bsp120I (PspOMI)
140
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...C T G C A↓G...3’
3’...G↑A C G T C...5’
#ER0611 3000 uSupplied with:10X Buffer O 2x1 ml10X Buffer Tango™ 1 ml
#ER0615 10000 uSupplied with:10X Buffer O 4x1 ml10X Buffer Tango™ 1 ml
#ER0612 5x3000 uSupplied with:10X Buffer O 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® PstI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer O at 37°C.
Storage BufferPstI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 200 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.15% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with PstI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm,CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Note• Conditions of high pH, low salt, high glycerol,
8% DMSO can cause star activity (Malyguine, E., et al., Gene, 8, 163-177, 1980).
• Surrounding sequences: the presence of adjacent runs of G-C base pairs confers sig-nificant resistance to cleavage (Armstrong, K. and Bauer, W.R., NAR, 10, 993-1007, 1982).
• 100% dUTP incorporation at the recognition site reduces PstI cleavage to 25% (Glenn, T.C., et al., Biotechniques, 17, 1086-1090, 1994).
• PstI will not cut AGCTGCAG when me-thylated by AluI methyltransferase.
PstI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 100 100 50-100 50-100
5’...G A C N↓N N G T C...3’
3’...C T G N N↑N C A G...5’
#ER1331 1000 uSupplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® PsyI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer B at 37°C.
Storage BufferPsyI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with PsyI, more than 60% of the DNA fragments can be ligated and more than 95% of these can be recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
PsyI (Tth111I) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 0-20 0-20 50-100 0-20
5’...R↓G A T C Y...3’
3’...Y C T A G↑R...5’
#ER1551 500 uSupplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® PsuI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer B at 37°C.
Storage BufferPsuI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with PsuI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
NoteHigh glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.
PsuI (BstYI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 20-50 0-20 0-20 50-100 0-20
141
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...C G A T↓C G...3’
3’...G C↑T A G C...5’
#ER0621 300 u#ER0622 1500 u
Both supplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® PvuI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer R at 37°C.
Storage BufferPvuI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with PvuI, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
PvuI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 50-100 100 50-100 100
RcaI Fermentas enzymes FastDigest® BspHI (PagI) and PagI (BspHI)
5’...C A G↓C T G...3’
3’...G T C↑G A C...5’
#ER0631 2500 uSupplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml
#ER0635 5000 u#ER0633 HC, 12500 u
Both supplied with:10X Buffer G 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® PvuII
Concentration10 u/µl50 u/µl, HC
Conditions for 100% Activity1X Buffer G at 37°C.
Storage BufferPvuII is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with PvuII, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NoteGreater than 15-fold overdigestion with PvuII may result in star activity.
PvuII Activity in Five Buffer System, %
* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
B G O R Tango 2X Tango 50-100* 100 20-50 50-100 20-50* 20-50*
5’...G T↓A C...3’
3’...C A↑T G...5’
#ER1121 1000 uSupplied with:10X Buffer Tango™ 1 ml
#ER1122 5000 uSupplied with:10X Buffer Tango™ 2x1 ml
Also available asFastDigest® RsaI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferRsaI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with RsaI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).
RsaI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 0-20 0-20 100 0-20
142
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
RsrII Fermentas enzymes FastDigest® RsrII (CpoI) and CpoI (RsrII)
5’...C A Y N N↓N N R T G...3’
3’...G T R N N↑N N Y A C...5’
#ER2001 200 u#ER2002 1000 u
Both supplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® MslI (RseI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer R at 37°C.
Storage BufferRseI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with RseI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG – no effect.EcoKI: may overlap – blocked (p.181).EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
RseI (MslI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 50-100 100 20-50 100
5’...G A G C T↓C...3’
3’...C↑T C G A G...5’
#ER1131 1200 u#ER1135 2000 u
Both supplied with:10X Buffer SacI 1 ml10X Buffer Tango™ 1 ml
#ER1132 5x1200 uSupplied with:10X Buffer SacI 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® SacI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer SacI at 37°C.
Storage BufferSacI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with SacI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Note• SacI is sensitive to cytosine methylation at
GAGmCTC but not GAGCTmC and insensi-tive to adenine methylation at GmAGCTC. AluI methyltransferase (AGmCT) can be used to block SacI.
• Supercoiled plasmids may require up to 5-fold more SacI for complete digestion than linear DNA.
• SacI is inhibited by common clinical anticoagulants found in some preparation of anticoagulated peripheral blood and bone marrow. Levels of EDTA and ACD (citric acid-sodium citrate-dextrose) in standard sample preparation have been shown to inhibit SacI. Three times the normal concentration for heparin is required to inhibit SacI. (Coad, J.E., et al., Inhibition of restriction endonu-cleases by common clinical anticoagulants, Anal. Biochem., 205, 368-369,1992).
SacI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 0-20 0-20 50-100 20-50
SacII Fermentas enzyme Cfr42I (SacII)
143
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
SapI Fermentas enzymes FastDigest® SapI (LguI) and LguI (SapI)
SanDI Fermentas enzyme FastDigest® SanDI (KflI)
5’...G↓T C G A C...3’
3’...C A G C T↑G...5’
#ER0641 1500 u#ER0645 2000 u
Both supplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
#ER0642 5x1500 uSupplied with:10X Buffer O 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® SalI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer O at 37°C.
Storage BufferSalI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C),100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with SalI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Note• Low salt, high glycerol (>5%) concentra-
tions, pH >8.0 or a large excess of enzyme may result in star activity.
• Supercoiled forms of pBR322 and pUC require 10-fold overdigestion with SalI to achieve complete digestion.
• Incubation at 25°C results in 50-75% activity.
SalI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 100 20-50 0-20 50-100
SauI Fermentas enzymes FastDigest® Bsu36I (Eco81I) and Eco81I (Bsu36I)
Sau96I Fermentas enzymes FastDigest® Sau96I (Cfr13I) and Cfr13I (Sau96I)
Sau3AI Fermentas enzymes FastDigest® Sau3AI (Bsp143I) and Bsp143I (Sau3AI); FastDigest® DpnI and DpnI (different cleavage position and different sensitivity to methylation); FastDigest® MboI and MboI (different sensitivity to methylation)
SbfI Fermentas enzymes FastDigest® SbfI (SdaI) and SdaI (SbfI)
5’...G C↓N G C...3’
3’...C G N↑C G...5’
#ER1641 200 u#ER1642 1000 u
Both supplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® Fnu4HI (SatI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer G at 37°C.
Storage BufferSatI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with SatI, more than 60% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – blocked (p.179).
SatI (Fnu4HI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 20-50 20-50 50-100 20-50
NoteAt least two copies of SatI recognition site are required for efficient cleavage.
144
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...A G T↓A C T...3’
3’...T C A↑T G A...5’
#ER0431 1000 uSupplied with:10X Buffer ScaI 1 ml
#ER0432 5000 uSupplied with:10X Buffer ScaI 2x1 ml
Also available asFastDigest® ScaI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer ScaI at 37°C.
Storage BufferScaI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with ScaI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – blocked (p.181).
Digestion of Agarose-embedded DNAMinimum 20 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded λ DNA in 16 hours.
Note• Conditions of low salt, high enzyme concen-
tration, glycerol concentration > 5% or pH > 8.0 may result in star activity.
• Supercoiled plasmids may require up to 20-fold more ScaI for complete digestion than linear DNAs.
ScaI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 0-20 0-20 0-20
Methylation EffectsDam, Dcm,CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
5’...G A G T C(N)5↓...3’
3’...C T C A G(N)5↑...5’
#ER1371 1000 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® MlyI (SchI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferSchI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with SchI, approxi-mately 90% of DNA fragments can be ligated and more than 90% of these can be recut.
Note• Greater than 10-fold overdigestion with SchI
may result in star activity.• SchI may remain associated with the cleaved
DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
SchI (MlyI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 0-20 0-20 100 0-20
ScrFI Fermentas enzymes FastDigest® ScrFI (Bme1390I) and Bme1390I (ScrFI)
5’...C C T G C A↓G G...3’
3’...G G↑A C G T C C...5’
#ER1191 300 u#ER1192 1500 u
Both supplied with:10X Buffer SdaI 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® SbfI (SdaI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer SdaI at 37°C.
Storage BufferSdaI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 5-fold overdigestion with SdaI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NoteGreater than 5-fold overdigestion with SdaI may result in star activity.
SdaI (SbfI) Activity in Five Buffer System, % B G O R Tango 2X Tango NR NR 0-20 0-20 NR 20-50
145
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...G C G A T↓C G C...3’
3’...C G C↑T A G C G...5’
#ER2091 1000 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® AsiSI (SfaAI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferSfaAI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with SfaAI, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded pJET1 DNA with inserted SfaAI recognition site in 16 hours.
SfaAI (AsiSI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 0-20 0-20 0-20 100 0-20
SecI Fermentas enzymes FastDigest® BsaJI (BseDI) and BseDI (BsaJI)
SexAI Fermentas enzyme FastDigest® SexAI (CsiI)
5’...G D G C H↓C...3’
3’...C↑H C G D G...5’
#ER0651 500 uSupplied with:10X Buffer SduI 1 ml
Also available asFastDigest® Bsp1286I (SduI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer SduI at 37°C.
Storage BufferSduI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with SduI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG – no effect.EcoKI, EcoBI: may overlap – effect not determined.
NoteLow salt, high glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.
SduI (Bsp1286I) Activity in Five Buffer System, %
* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
B G O R Tango 2X Tango NR 50-100* 50-100 0-20 NR NR
SfaNI Fermentas enzymes FastDigest® SfaNI (BmsI) and LweI (SfaNI)
SfcI Fermentas enzymes FastDigest® SfcI (BfmI) and BfmI (SfcI)
SfeI Fermentas enzymes FastDigest® SfcI (BfmI) and BfmI (SfcI)
146
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
SfoI Fermentas enzymes FastDigest® EheI and EheI (SfoI); Fermentas enzyme FastDigest® SspDI (KasI) (different cleavage position)
SfuI Fermentas enzymes FastDigest® Bsp119I and Bsp119I (BstBI)
SgfI Fermentas enzymes FastDigest® AsiSI (SfaAI) and SfaAI (AsiSI)
5’...G G C C N N N N↓N G G C C...3’
3’...C C G G N↑N N N N C C G G...5’
#ER1821 1000 uSupplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® SfiI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer G at 50°C.
Storage BufferSfiI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 300 mM NaCl, 10 mM MgCl2, 1 mM DTT, 0.15% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with SfiI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded Adenovirus-2 DNA in 16 hours.
Note• Incubation at 37°C results in 10% activity. • SfiI cleavage is impaired by overlapping dcm
methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
• For cleavage with SfiI at least two copies of its recognition sequence are required. The two sites can be on either the same or different DNA molecules. (Wertzell, L.M. et al., J. Mol. Biol., 248, 581-595, 1995.) Therefore also an oligonucleotide harboring a SfiI recognition site can be supplemented.
SfiI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 100 20-50 0-20 100 0-20
Note• DNA methylated by Dcm or CpG methyltrans-
ferases will be a substrate for SgeI.• Greater than 3-fold overdigestion with SgeI
may result in nonspecific cleavage.• At least two copies of SgeI recognition site
are required for an efficient cleavage.• Amount of the enzyme required for complete
digestion of methylated DNA depends on the number of SgeI recognition sites. DNA cleavage products generated by target site cleavage facilitate the nonspecific cleavage by SgeI. Therefore, optimization of enzyme amount is recommended for DNA cleavage.
• pBR322 DNA isolated from E.coli dcm+ strain (#SD0041) can be used as a DNA cleavage efficiency control. SgeI cleaves all six dcm-methylated targets on pBR322 DNA.
Concentration3 u/µl
Conditions for 100% Activity1X Buffer SgeI at 37°C.
Storage BufferSgeI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA and 50% glycerol.
Ligation and RecleavageAfter a 2-fold overdigestion with SgeI, more than 80% of the DNA fragments can be ligated and recut..
Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm: always cleaves DNA methylated by Dcm methyltransferase (p.177).CpG: cleaves targets overlapping with CpG methylated sequences (p.179).
5’... m5C N N G (N)9 ↓...3’
3’... G N N C (N)13↑...5’** SgeI cleaves DNA targets containing 5-methylcytosine on
one or both DNA strands
#ER2211 250 uSupplied with:10X Buffer SgeI 1 ml
SgeI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 NR NR NR
Digestion of Agarose-embedded DNAA minimum of 3 units of the enzyme is required for complete digestion of 1 µg of agarose-embedded lambda DNA in 16 hours.
147
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
SinI Fermentas enzymes FastDigest® AvaII (Eco47I) and Eco47I (AvaII)
5’...G G↓C G C G C C...3’
3’...C C G C G C↑G G...5’
#ER1891 300 u#ER1892 1500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® AscI (SgsI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferSgsI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with SgsI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
SgsI (AscI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 50-100 100 50-100
5’...C C C↓G G G...3’
3’...G G G↑C C C...5’
#ER0661 1200 u#ER0665 2000 u
Both supplied with:10X Buffer Tango™ 1 ml
#ER0662 5x1200 u#ER0663 HC, 6000 u
Both supplied with:10X Buffer Tango™ 2x1 ml
Also available asFastDigest® SmaI
Concentration10 u/µl50 u/µl, HC
Conditions for 100% Activity 1X Buffer Tango™ at 30°C.
Storage BufferSmaI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with SmaI, more than 90% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Note• Incubation at 37°C results in 50% activity.
SmaI has a half-life of 15 min at 37°C.• Incubation at 25°C results in 100% activity.• SmaI needs K+ to work for activity.
SmaI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 0-20 0-20 0-20 100 0-20
5’...C G↓T C G A C G...3’
3’...G C A G C T↑G C...5’
#ER2031 200 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Concentration5 u/µl
Conditions for 100% Activity1X Buffer R at 37°C.
Storage BufferSgrDI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 30-fold overdigestion with SgrDI, more than 80% of the DNA fragments can be ligated. More than 95% of these can be recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NoteLow salt concentration or large excess of the enzyme may result in star activity.
SgrDI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 100 NR 100
148
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
SmlI Fermentas enzyme SmoI (SmlI)
5’...A T T T↓A A A T...3’
3’...T A A A↑T T T A...5’
#ER1241 1000 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® SwaI (SmiI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer O at 30°C.
Storage BufferSmiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with SmiI, more than 80% of the DNA fragments can be ligated and more than 95% of these can be recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded Adenovirus-2 DNA in 16 hours.
NoteIncubation at 37°C results in 70% activity.
SmiI (SwaI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 100 20-50 0-20 20-50
5’...C↓T Y R A G...3’
3’...G A R Y T↑C...5’
#ER1981 200 uSupplied with:10X Buffer Tango™ 1 ml
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 55°C.
Storage BufferSmoI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with SmoI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam: may overlap – cleavage impaired (p.176).Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Note• Incubation at 37°C results in 10% activity.• SmoI cleavage is impaired by overlapping dam
methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
• Low salt, high glycerol (>5%) concentra-tions, pH >8.0 or a large excess of enzyme may result in star activity.
SmoI (SmlI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 0-20 20-50 100 20-50
5’...C C C G C(N)4↓...3’
3’...G G G C G(N)6↑...5’
#ER1691 50 uSupplied with:10X Buffer Tango™ 1 ml
Concentration2 u/µl
Conditions for 100% Activity 1X Buffer Tango™ at 37°C.
Storage BufferSmuI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with SmuI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
NoteGreater than 40-fold overdigestion with SmuI may result in star activity.
SmuI (FauI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 20-50 100 20-50
149
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...C↓C G C...3’
3’...G G C↑G...5’
#ER1791 200 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest®AciI (SsiI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer O at 37°C.
Storage BufferSsiI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with SsiI, approxi-mately 95% of the DNA fragments can be ligated. No more than 50% of these can be recut due to asymmetric recognition sequence of SsiI.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
NoteLow salt, high glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.
SsiI (AciI) Activity in Five Buffer System, % B G O R Tango 2X Tango NR 20-50 100 50-100 NR 100
5’...A A T↓A T T...3’
3’...T T A↑T A A...5’
#ER0771 500 u#ER0772 2500 u
Both supplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® SspI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer G at 37°C.
Storage BufferSspI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with SspI, more than 90% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NoteGreater than 15-fold overdigestion with SspI may result in star activity.
SspI Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 0-20 50-100 100 20-50
SnaI Fermentas enzymes FastDigest® BstZ17I (Bst1107I) and Bst1107I (BstZ17I)
SnaBI Fermentas enzymes FastDigest® SnaBI (Eco105I) and Eco105I (SnaBI)
SpeI Fermentas enzymes FastDigest® SpeI (BcuI) and BcuI (SpeI)
SphI Fermentas enzymes FastDigest® SphI (PaeI) and PaeI (SphI)
SplI Fermentas enzymes FastDigest® BsiWI (Pfl23II) and Pfl23II (BsiWI)
Sse232I Fermentas enzymes FastDigest® MreI and MreI (Sse232I)
Sse8387I Fermentas enzymes FastDigest® SbfI (SdaI) and SdaI (SbfI)
SspBI Fermentas enzymes FastDigest® Bsp1407I and Bsp1407I (BsrGI)
150
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
StuI Fermentas enzymes FastDigest® StuI (Eco147I) and Eco147I (StuI)
StyI Fermentas enzymes FastDigest® StyI (Eco130I) and Eco130I (StyI)
StyD4I Fermentas enzymes FastDigest® ScrFI (Bme1390I) and Bme1390I (ScrFI) (different cleavage position)
SwaI Fermentas enzymes FastDigest® SwaI (SmiI) and SmiI (SwaI)
5’...A C N↓G T...3’
3’...T G↑N C A...5’
#ER1361 200 u#ER1362 1000 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® TaaI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 65°C.To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.
Storage BufferTaaI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and Recleavage After 50-fold overdigestion with TaaI, approxi-mately 90% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI: may overlap – effect not determined.
NoteIncubation at 37°C results in 10% activity.
TaaI (HpyCH4III) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 50-100 100 100
5’...G↓G C G C C...3’
3’...C C G C G↑G...5’
#ER2191 250 uSupplied with:10X Buffer Tango™ 1 ml
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferSspDI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter a 50-fold overdigestion with SspDI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
SspDI (KasI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 0-20 NR 0-20 100 20-50
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer R at 65°C.To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.
Storage BufferTaiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with TaiI, more than 95% of DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not determined.
NoteIncubation at 37°C results in less than 10% activity.
Tail (MaeII*) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 100 100 50-100
* Unlike MaeII, TaiI produces DNA fragments with a 4-base 3’-extension
5’... A C G T↓...3’
3’...↑T G C A ...5’
#ER1141 400 u#ER1142 2000 u
Both supplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
FastDigest® TaiI
151
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...↓A A T T ...3’
3’... T T A A↑...5’
#ER1351 1000 uSupplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
#ER1352 5000 uSupplied with:10X Buffer B 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® Tsp509I (TasI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer B at 65°C.To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.
Storage BufferTasI is supplied in:10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with TasI, more than 95% of DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – blocked (p.181).
NoteIncubation at 37°C results in less than 10% activity.
TasI (Tsp509I) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 20-50 0-20 20-50 0-20
5’...T↓C G A...3’
3’...A G C↑T...5’
#ER0671 3000 uSupplied with:10X Buffer TaqI 2x1 ml10X Buffer Tango™ 1 ml
#ER0672 5x3000 uSupplied with:10X Buffer TaqI 4x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® TaqI
Concentration10 u/µl,
Conditions for 100% Activity1X Buffer TaqI at 65°C.To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.
Storage BufferTaqI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with TaqI, more than 95% of DNA fragments can be ligated and recut.
Methylation EffectsDam: may overlap – blocked (p.176).Dcm, CpG, EcoKI, EcoBI – no effect.
Note• TaqI is blocked by overlapping dam methyla-
tion. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
• Incubation at 37°C results in 10% activity. We recommend using TaqI, HC (#ER0673) at 37°C.
TaqI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 20-50 20-50 20-50 20-50
5’...W↓G T A C W...3’
3’...W C A T G↑W...5’
#ER1291 100 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® TatI
Concentration5 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 65°C.To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.
Storage BufferTatI is supplied in:10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 2-fold overdigestion with TatI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Note• Incubation at 37°C results in 20% activity.• Greater than 5-fold overdigestion with TatI
may result in star activity.
* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).
TatI Activity in Five Buffer System, % B G O R Tango 2X Tango NR 50-100* 20-50 20-50 100* 0-20
152
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
TfiI Fermentas enzymes FastDigest® TfiI (PfeI) and PfeI (TfiI)
TliI Fermentas enzymes FastDigest® XhoI and XhoI
5’...T↓T A A ...3’
3’...A A T↑T...5’
#ER0981 300 u#ER0982 1500 u#ER0983 HC, 1500 u
All supplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® Tru1I
Concentration10 u/µl50 u/µl, HC
Conditions for 100% Activity1X Buffer R at 65°C.To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.
Storage BufferTru1I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Tru1I, more than 90% of DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – blocked (p.181).
NoteIncubation at 37°C results in 10% activity. We recommend using Tru1I, HC (#ER0983) at 37°C.
Tru1I (MseI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 100 50-100 100
5’...G C S G↓C...3’
3’...C↑G S C G...5’
#ER1651 50 u#ER1652 250 u
Both supplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® TauI
Concentration3 u/µl
Conditions for 100% Activity1X Buffer B at 55°C.
Storage BufferTauI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with TauI, more than 90% of DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).
Note• Incubation at 37°C results in 30% activity.• TauI may remain associated with the cleaved
DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
TauI Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 0-20 0-20 20-50 0-20
Tru9I Fermentas enzymes FastDigest® Tru1I and Tru1I (MseI); Fermentas enzyme FastDigest® MseI (SaqAI)
153
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...T A R C C A (N)11↓...3’
3’...A T Y G G T (N) 9↑...5’
#ER1991 50 uSupplied with:10X Buffer G 1 ml50X SAM 0.1 ml10X Buffer Tango™ 1 ml
Concentration3 u/µl
Conditions for 100% Activity1X Buffer G at 55°C.SAM 0.05 mM.
Storage Buffer TsoI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with TsoI, approxi-mately 80% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.
Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – effect not determined.
Note• Incubation at 37° results in 10% activity.• TsoI requires only Mg2+ for its activity, but
is stimulated by S-adenosylmethionine. 0.05 mM S-adenosylmethionine gives a 2-fold increase in TsoI activity. Still, com-plete cleavage of some substrates with TsoI is difficult to achieve.
• TsoI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.
• Low salt, high glycerol (>5%) concentra-tions, pH >8.0 or a large excess of enzyme may result in star activity.
• TsoI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
TsoI Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM
NR 100 50-100 0-20 50-100 20-50
Tsp4CI Fermentas enzymes FastDigest® TaaI and TaaI (HpyCH4III)
Tsp45I Fermentas enzymes FastDigest® NmuCI and NmuCI (Tsp45I)
Tsp509I Fermentas enzymes FastDigest® Tsp509I (TasI) and TasI (Tsp509I)
TspMI Fermentas enzymes Cfr9I (XmaI); FastDigest® SmaI and SmaI (different cleavage position)
TspRI Fermentas enzymes FastDigest® TspRI (TscAI) and TscAI (TspRI)
5’... N N C A S T G N N↓...3’
3’...↑N N G T S A C N N ...5’
#ER2101 1000 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® TspRI (TscAI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 65°C.To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.
Storage BufferTscAI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with TscAI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG – no effect.EcoKI, EcoBI: may overlap – effect not determined.
Note• Incubation at 37°C results in less than 5%
activity.• Low salt, high glycerol (>5%) concentra-
tions, pH >8.0 or a large excess of enzyme may result in star activity.
• TscAI produces DNA fragments with a 9-base 3’-extension. To avoid atypical DNA band patterns, use 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
TscAI (TspRI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 20-50 100 20-50
154
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Tth111I Fermentas enzymes FastDigest® PsyI and PsyI (Tth111I)
5’...↓ 8(N)CAC(N)6TCC(N)12↓ ...3’
3’...↑13(N)GTG(N)6AGG(N) 7↑...5’
#ER1911 100 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Concentration5 u/µl
Conditions for 100% Activity1X Buffer R at 37°C.
Storage BufferTstI is supplied in:10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with TstI, more than 80% of DNA fragments can be ligated and recut.
Methylation EffectsDam: may overlap – blocked (p.176).Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
Note• TstI is blocked by overlapping dam methyla-
tion. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
• The presence of S-adenosylmethionine in a reaction mixture results in incomplete
cleavage with TstI.• Greater than 10-fold overdigestion with TstI
may result in star activity.• TstI may remain associated with the cleaved
DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical
DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
TstI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 100 0-20 100
VpaK11BI Fermentas enzymes FastDigest® AvaII (Eco47I) and Eco47I (AvaII)
5’...A T↓T A A T...3’
3’...T A A T↑T A...5’
#ER0911 1000 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
#ER0912 5000 uSupplied with:10X Buffer O 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® AseI (VspI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer O at 37°C.
Storage BufferVspI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with VspI, more than 95% of DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
VspI (AseI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 100 20-50 100 100
5’...C C A N N N N↓N T G G...3’
3’...G G T N↑N N N N A C C...5’
#ER0711 400 u#ER0712 2000 u
Both supplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® PflMI (Van91I)
Concentration10 u/µl
Conditions for 100% Activity 1X Buffer R at 37°C.
Storage BufferVan91I is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with Van91I, more than 90% of DNA fragments can be ligated and recut.
Methylation EffectsDam, CpG, EcoKI, EcoBI – no effect.Dcm: may overlap – blocked (p.177).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NoteVan91I is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
Van91I (PflMI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 50-100 100 20-50 50-100
155
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
5’...R↓A A T T Y...3’
3’...Y T T A A↑R...5’
#ER1381 500 uSupplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® XapI
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferXapI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 10-fold overdigestion with XapI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NoteGreater than 15-fold overdigestion with XapI may result in star activity.
XapI (ApoI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 100 0-20 0-20 100 20-50
5’...T C T A G A...3’
3’...A G A T C T...5’
#ER0681 1500 uSupplied with:10X Buffer Tango™ 1 ml
#ER0685 3000 u#ER0682 5x1500 u#ER0683 HC, 7500 u
All supplied with:10X Buffer Tango™ 2x1 ml
Also available asFastDigest® XbaI
Concentration10 u/µl50 u/µl, HC
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferXbaI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with XbaI, more than 95% of DNA fragments can be ligated and recut.
Methylation EffectsDam: may overlap – blocked (p.176).Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NoteXbaI is blocked by overlapping dam methy-lation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).
XbaI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 0-20 100 50-100
5’...C C T N N↓N N N A G G...3’
3’...G G A N N N↑N N T C C...5’
#ER1301 1000 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® EcoNI (XagI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer R at 37°C.
Storage BufferXagI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with XagI, approxi-mately 80% of DNA fragments can be ligated and more than 95% of these fragments can be recut.
Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
XagI (EcoNI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 50-100 100 20-50 50-100
156
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...R C A T G↓Y...3’
3’...Y↑G T A C R...5’
#ER1471 500 u#ER1472 2500 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® NspI (XceI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferXceI is supplied in:10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with XceI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
XceI (NspI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 0-20 0-20 0-20 100 0-20
5’...C↓T C G A G...3’
3’...G A G C T↑C...5’
#ER0691 2000 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
#ER0695 5000 u#ER0692 5x2000 u#ER0693 HC, 10000 u
All supplied with:10X Buffer R 2x1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® XhoI
Concentration10 u/µl50 u/µl, HC
Conditions for 100% Activity1X Buffer R at 37°C.
Storage BufferXhoI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with XhoI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – cleavage impaired (p.178).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
NoteSupercoiled plasmids may require up to 5-fold more XhoI for complete digestion than linear DNA.
XhoI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 50-100 100 20-50 100
XhoII Fermentas enzymes FastDigest® PsuI and PsuI (BstYI)
XmaI Fermentas enzymes Cfr9I (XmaI); FastDigest® SmaI and SmaI (different cleavage position)
XmaIII Fermentas enzymes FastDigest® EagI (Eco52I) and Eco52I (EagI)
XmaCI Fermentas enzymes Cfr9I (XmaI); FastDigest® SmaI and SmaI (different cleavage position)
5’...C↓C T A G G...3’
3’...G G A T C↑C...5’
#ER1561 200 u#ER1562 1000 u
Both supplied with:10X Buffer Tango™ 1 ml
Also available asFastDigest® AvrII (XmaJI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™ at 37°C.
Storage BufferXmaJI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with XmaJI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded λ DNA in 16 hours.
XmaJI (AvrII) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 50-100 50-100 100 50-100
157
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
XmnI Fermentas enzymes FastDigest® PdmI and PdmI (XmnI)
XspI Fermentas enzymes FastDigest® BfaI (FspBI) and FspBI (BfaI)
ZraI Fermentas enzymes FastDigest® AatII and AatII (different cleavage position)
Nicking Enzymes
5’...G T↓M K A C...3’
3’...C A K M↑T G...5’
#ER1481 400 u#ER1482 2000 u
Both supplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml
Also available asFastDigest® AccI (XmiI)
Concentration10 u/µl
Conditions for 100% Activity1X Buffer B at 37°C.
Storage BufferXmiI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with XmiI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – blocked (p.179).
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.
XmiI (AccI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 0-20 0-20 0-20 50-100 20-50
5’...C C T N A G C...3’
3’...G G A N T↑C G...5’
#ER1681 1000 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Concentration5 u/µl
Conditions for 100% Activity1X Buffer R: 10 mM Tris-HCl (pH 8.5 at 37°C), 10 mM MgCl2, 100 mM KCl, 0.1 mg/ml BSA.Incubate at 37°C.
Storage BufferNb.Bpu10I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA, 50% glycerol.
Nicking and Cleavage• Incubation of 10 u of enzyme with 1 µg pUC19
DNA (lacking the recognition sequence of Bpu10I) for 1 h at 37°C in 50 µl reaction buffer results in <10% conversion to circular form.
• Incubation of 1 u of enzyme with 1 µg pBR322 DNA for 1 h at 37°C in 50 µl reaction buffer results in <5% conversion to linear form.
Applications• Production of single-stranded circular DNA
from supercoiled double-stranded plasmids in vitro with subsequent use in DNA sequenc-ing, site-specific mutagenesis, etc.
• Creation of nested deletions. • Vector preparation for ligation independent
cloning method.• Preparations of covalently closed, double-
stranded linear DNA molecules.
NoteNb.Bpu10I may remain associated with the cleaved DNA. This may cause DNA band shift-ing during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
Nb.Bpu10I Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 20-50 100 20-50 50-100
158
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
5’...G A A T G C...3’
3’...C T T A C↑G...5’
#ER2051 1000 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml
Concentration10 u/µl
Conditions for 100% Activity1X Buffer O:50 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 100 mM NaCl and 0.1 mg/ml BSA.Incubate at 37°C.
Storage BufferNb.Mva1269I is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA, 50% glycerol.
Nicking and Cleavage• Incubation of 10 u of enzyme with 1 µg
pUC19 DNA (lacking the recognition sequence of Mva1269I) for 16 h at 37°C in 50 µl reaction buffer results in <3% conversion to circular form.
• Incubation of 10 u of enzyme with 1 µg pBR322 DNA for 16 h at 37°C in 50 µl reaction buffer results in <1% conversion to linear form.
Applications• Production of single-stranded circular DNA
from supercoiled double-stranded plasmids in vitro with subsequent use in DNA sequenc-ing, site-specific mutagenesis, etc.
• Creation of nested deletions. • Vector preparation for ligation independent
cloning method.• Preparations of covalently closed, double-
stranded linear DNA molecules.
Nb.Mva1269I Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 100 20-50 50-100
5’...C C↓T N A G C...3’
3’...G G A N T C G...5’
#ER2011 1000 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml
Concentration5 u/µl
Conditions for 100% Activity 1X Buffer R: 10 mM Tris-HCl (pH 8.5 at 37°C), 10 mM MgCl2, 100 mM KCl, 0.1 mg/ml BSA. Incubate at 37°C.
Storage BufferNt.Bpu10I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA, 50% glycerol.
Nicking and Cleavage• Incubation of 20 u of enzyme with 1 µg pUC19
DNA (lacking the recognition sequence of Bpu10I) for 1 h at 37°C in 50 µl reaction buffer results in <3% conversion to circular form.
• No detectable conversion of pBR322 DNA (1 µg) to linear form was observed after incu-bation with 20 u of Nt.Bpu10I for 1 h at 37°C in 50 µl reaction buffer.
Applications• Production of single-stranded circular DNA
from supercoiled double-stranded plasmids in vitro with subsequent use in DNA sequenc-ing, site-specific mutagenesis, etc.
• Creation of nested deletions. • Vector preparation for ligation independent
cloning method.• Preparations of covalently closed, double-
stranded linear DNA molecules.
NoteNt.Bpu10I may remain associated with the cleaved DNA. This may cause DNA band shift-ing during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
Nt.Bpu10I Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 100 100 0-20 50-100
159
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
I-SceI is a site-specific homing enzyme encoded by a mitochondrial intron of Saccharomyces cerevisiae (1, 2). Intron-encoded endonucleases are proteins that promote the first step in mobility of the intron at the DNA level. They recognize and cleave an intronless allele of their cognate gene to insert a copy of the intron by a double-strand-break repair mechanism that results in the recipient allele also becoming intron-plus (3-5). Homing endonucleases recognize long, 14-40 base pairs sequences and are, therefore, extremely rare-cutting enzymes. They allow the introduction of a single or several double-strand breaks into complex genomes. This capability makes these enzymes powerful tools in high-resolution physical mapping, genome organization analysis, gene cloning, site-directed recombination and double-strand-break repair studies in diverse biological systems (4, 6).
Homing Enzyme
References1. Colleaux, L., et al., Recognition and cleavage site of the
intron-encoded omega transposase, Proc. Natl. Acad.Sci. U.S.A.,85, 6022-6026, 1988.
2. Monteihet, C., et al., Purification and characterization of the in vitro activity of I-SceI, a novel and highly specific endonuclease encoded by a group I intron, Nucleic Acids Res., 18, 1407-1413, 1990.
3. Dujon, B., Group I introns as mobile genetic elements: facts and mechanistic speculations – review, Gene, 82, 91-114, 1989.
4. Belfort, M., Roberts R.J., Homing endonucleases: keeping the house in order, Nucleic Acids Res., 25, 3379-3388, 1997.
5. Chevalier, B.S., Stoddard, B.L., Homing endonucleases:structural and functional insight into the catalysis of intron/intein mobility, Nucleic Acids Res., 29, 3757-3774, 2001.
6. Jasin, M., Genetic manipulation of genomes with rare-cutting endonucleases, Trends in Genetics, 12, 224-228, 1996.
Digestion of the Agarose-embedded DNA with I-SceI1. Immerse an agarose plug in 50-100 µl of the 1X Tango™ buffer without Mg-acetate* (supplied with the
enzyme). The volume of the buffer should be sufficient to completely cover the plug.2. Add 20 u of the enzyme.3. Incubate 2 hours on ice.4. Add 1/10 volume of the 100 mM Mg-acetate solution (supplied with the enzyme).5. Incubate at 37°C for 1 hour. Note* Diffusion of the enzyme in the absence of Mg-acetate prior to digestion is necessary, because I-SceI is unstable in the
presence of Mg2+ ions.
5’...T A G G G A T A A↓C A G G G T A A T...3’
3’...A T C C C↑T A T T G T C C C A T T A...5’
#ER1771 250 uSupplied with:10X Buffer Tango™ 1 ml10X Buffer Tango™ (without Mg-acetate) 1 ml100mM Mg-acetate 1 ml
Concentration10 u/µl
Conditions for 100% Activity1X Buffer Tango™:33 mM Tris-acetate (pH 7.9 at 37°C), 10 mM Mg-acetate, 66 mM K-acetate and 0.1 mg/ml BSA. Incubate at 37°C.
Storage BufferI-SceI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 500 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% glycerol.
Ligation and RecleavageAfter 50-fold overdigestion with I-SceI, more than 95% of the DNA fragments can be ligated and recut.
Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.
Digestion of Agarose-embedded DNAMinimum 20 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded pUC-I-SceI DNA in 1 hour (see the protocol below).
Note• Homing enzymes do not have stringently
defined recognition sequences. They can tolerate minor sequence changes, which only partially affect the cleavage reaction.
• I-SceI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electro-phoresis.
• Assayed using pUC-I-SceI DNA.
I-SceI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 50-100 50-100 100 50-100
160
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Protocols and Recommendations
1.5. DNA digestionWe recommend digesting 0.2-1.5 µg DNA with a 2-fold to 10-fold excess of enzyme in a total volume of 20 µl . A typical restriction enzyme digestion protocol is below.1. Add the following reaction components in
the order indicated:Water, nuclease-free (#R0581) 16-16.5 µl 10X recommended buffer for restriction enzyme 2 µl Substrate DNA 1 µl (~1 µg)Restriction enzyme 0.5-1 µl (5-10 u)Total volume 20 µl
2. Mix gently and spin down briefly.3. Incubate at the optimal reaction tempera-
ture for 1-16 hours.Note• The digestion reaction may be scaled either up or down.• Some enzymes require additional components to obtain
the stated activity. In these cases, add the required addi-tive and adjust the volume of water appropriately.
• See Tables 1.5 and 1.6 for restriction enzyme buffer composition and reaction conditions, respectively.
1.6. Digestion of PCR productsThe most convenient option for digestion of PCR-amplified DNA is the addition of a restric-tion enzyme directly to the reaction tube after completion of PCR. The majority of Fermentas restriction enzymes are active in Fermentas PCR buffers. However, digestion of PCR products in the am-plification mixture is often inefficient. Therefore, PCR reaction mixture should not make more than 1/3 volume of digestion reaction mixture to avoid inhibitory effects. 1. Add the following reaction components in
the order indicated:PCR reaction mixture 10 µl (~0.1-0.5 µg
of DNA)
Water, nuclease-free (#R0581) 16-17 µl
10X recommended buffer for restriction enzyme 2 µl *
Restriction enzyme 1-2 µl (10-20 u)
Total volume 30 µl
* Only 2 µl of 10X reaction buffer is required for unpurified PCR product in a 30 µl reaction volume.
2. Mix gently and spin down briefly.3. Incubate at the optimal reaction tempera-
ture for 1-16 hours.Note• For cloning applications, purification of PCR products prior
to digestion is necessary to remove the active thermophilic DNA polymerase present in the PCR mixture. DNA polyme-rases may alter the ends of the cleaved DNA and reduce the yield of ligation.
• If the restriction enzyme requires special additives (e.g., SAM), reduce the amount of water appropriately.
• If cleavage of the PCR product is inefficient purify the PCR product with the GeneJET™ PCR Purification Kit (#K0702) prior to digestion.
• After digestion, gel-purify the PCR product with the GeneJET™ Gel Extraction Kit (#K0692) or Silica Bead DNA Gel Extraction Kit (#K0513) to remove short DNA fragments which compete with the insert in a ligation reaction.
1.7. Setting up double digestion
1.7.1. Double digestion with FastDi-gest® enzymesFor protocols and recommendations for FastDi-gest® restriction enzymes see p.70.
1.7.2. DoubleDigest™ EngineDoubleDigest™ engine (www.fermentas/doubl-edigest.com) is a web tool for finding informa-tion on buffer and reaction conditions for double digests. Simply select two restriction enzymes required for digestion, submit the query and follow the recommendations.
1.7.3. Double digestion in the univer-sal Tango™ BufferUse Table 1.8 (p.168) to set up the optimal condi-tions for your double digestion reactions in the universal Tango™ buffer.1. Determine the concentration of Tango™ buffer
recommended for each restriction enzyme.2. If the recommended concentration of Tango™
buffer is the same for both enzymes, the digests can be performed at the same time.
3. If the two restriction enzymes require different concentrations of Tango™ buffer, perform the digestions sequentially. Set-up the first digestion reaction (including both enzymes) in 1X Tango™ buffer, then add an additional aliquot of the 10X Tango™ buffer (1/8 of initial reaction volume) to attain a final 2X Tango™ buffer concentration to optimize conditions for the second enzyme.
NoteIf both 1X and the 2X concentrations of Tango™ buffer are suitable for the double digest, use the 2X concentrated buffer to reduce probability of star activity.
1.7.4. Double digestion in Five Buffer SystemTable 1.6 (p.163) presents the activities of Fermentas restriction enzymes in the Five Buffer System.1. Determine which color-coded buffers are
recommended for each enzyme.2. If possible, use the buffer in which both
enzymes have 100% activity.3. If this is not possible, choose the buffer in
which both enzymes maintain at least 20% of their activity. Increase the amount of the enzymes in your digest according to their activity in that buffer.
NoteFor enzymes that are prone to relaxation of target specificity use a buffer in which they do not exhibit star activity.
1.7.5. Digestion with enzymes having different temperature optimumsFor double digestion with enzymes working at different temperatures perform sequential DNA cleavage. The optimal reaction temperature for each restriction enzyme is indicated both in the product description and the Certificate of Analysis. Information about the activity of mesophilic and thermophilic restriction enzymes at 37°C is given in Table 1.7 on p.167.
1.7.6. Sequential digestionIf a double digest is not possible due to buffer incompatibility or star activity, perform sequential digestions in the optimal buffer for each enzyme.1. Digest the DNA with the first restriction
enzyme in its optimal buffer.2. Purify the digested DNA by spin column or
chloroform extraction and ethanol precipitation.3. Digest the DNA with the second restriction
enzyme in its optimal buffer.
1.8. Stability during prolonged incubationThe stability of restriction enzymes in a reaction mixture depends on the nature of the enzyme, the buffer composition and the incubation temperature.If a restriction enzyme retains its activity in the reaction mixture for more than one hour, DNA can be digested with less enzyme in a pro-longed incubation period. The exact quantities of enzymes sufficient for overnight digestion are listed in the Table 1.6 on p.163.
1.9. Dilution of restriction enzymesDilution Buffer for Restriction Enzymes (#B19) is available from Fermentas for applications that require diluted enzymes.Enzymes diluted in this buffer retain 50-100% activity after storage for one month at -20°C.
1.10. Partial digestion of DNACertain cloning experiments may require incom-plete DNA cleavage. Such partial digestion of the DNA can be achieved by using the following conditions: • suboptimal concentration of the restriction
enzyme in the reaction mixture, • short incubation time, • incubation at a suboptimal temperature. For certain targets, partial cleavage of the desired DNA site is inefficient due to site prefer-ences of restriction enzymes (see Site Prefer-ences by Restriction Endonucleases on p.173).
(continued on next page)
161
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Protocols and Recommendations p.160
1.11. Digestion of agarose-embedded DNA
1. Embed the substrate DNA in 1% low melting temperature agarose, 1 µg / 30 µl.
2. Prepare ~30 µl agarose plugs with a GelSy-ringe® or similar agarose dispensing system.
3. Equilibrate the plug in 100 µl of the appropri-ate 1X restriction enzyme buffer for 15 min.
4. Place the plug in 100 µl of fresh 1X buffer containing the restriction enzyme (see Table 1.9 on p.170 for recommended quantities of enzymes).
5. Incubate at 30-37°C for mesophilic enzymes or at 50-55°C for thermophilic enzymes for 4-16 hours.
1.12. Inactivation of restriction enzymesInactivation of restriction enzymes following a digestion reaction is often required for down-stream applications. Thermal inactivation is a convenient method used to terminate enzyme activity. The majority of restriction enzymes can be heat-inactivated at 65°C or 80°C in 20 min. Information on susceptibility of Fermentas restriction enzymes to thermal inactivation is listed in the Table 1.6 on p.163), as well as in product descriptions and certificates of analysis.All known restriction enzymes with exception of BfiI, BmuI and BmrI require Mg2+ for DNA cleavage. Thus, addition of EDTA (20 mM final concentration) to the reaction mixture is an alternative method that can be used to halt digestion. EDTA is generally not compat-ible with most of downstream applications, therefore purification of the digested DNA using GeneJET™ PCR Purification Kit (#K0702) or by chloroform extraction (see p.358) is recom-mended.
162
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
an oligonucleotide (also supplied with the enzyme), and Esp3I requires DTT.The following enzymes require unique buffers for optimal digestion: AarI, AjiI (BmgBI), BamHI, BfuI (BciVI), Bpu10I, BseXI (BbvI), Bsp143I (Sau3AI), Cfr9I (XmaI), Cfr10I (BsrFI), Eam1105I (AhdI), Ecl136II (EcoICRI), Eco52I (EagI), EcoRI, KpnI, Lsp1109I (BbvI), PacI, PasI, Ppu21I (BsaAI), SacI, ScaI, SdaI (SbfI), SduI (Bsp1286I), SgeI and TaqI. The compositions of these unique buffers are listed in Table 1.5 as well as in of restriction enzymes Certificates of Analysis.
All Fermentas restriction enzyme buffers should be stored at -20°C.
Reaction Conditions
Reaction BuffersOur Five Buffer System ensures the optimum reaction conditions for each restriction enzyme. This system consists of 10X B (blue), G (green), O (orange), R (red) and Tango™ (yellow) buffers (Table 1.6). All restriction enzymes are supplied in color-coded tubes to indicate the recom-mended reaction buffer. The recommended buffer and/or the universal Tango™ buffer, witch has been designed for double digestions of DNA, are supplied with each enzyme. Restriction enzyme buffers are also available separately. see Table 1.5 below for ordering information. For more information on double digestion see p.160 or visit the Fermentas DoubleDigest™ engine at www.fermentas.com/doubledigest.
To ensure consistent enzyme performance, Fermentas restriction enzyme buffers contain BSA, which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations. Multiple freeze-thaw cycles of the buffers will not cause BSA precipitation.
Fermentas restriction enzymes exhibit 100% of their certified activity in the recommended buffer. However, some enzymes require additives to achieve 100% activity. For example, AjuI, AlfI, BdaI, BplI, BseMII (BspCNI), Eco57I (AcuI), Eco57MI, FaqI (BsmFI), Hin4I, TsoI require S-adenosylmethionine, which is supplied with the enzyme, while AarI and BveI (BspMI) require
Table 1.5. Reaction buffers for restriction enzymes.
10X bufferCat.
#
1X buffer compositionpH, at
37°CQuantity,
ml
Tris-HCl, mM
Tris-acetate,
mM
Bis-Tris Propane-HCl,
mM
MgCl2,mM
NaCl, mM
KCl, mM
Mg-acetate,
mM
K-acetate, mM
Sodium glutamate,
mM
Triton X-100,
%
EDTA, mM
DTT, mM
Gly cerol,
%
BSA, mg/ml
Five Buffer System
10X Buffer B BB5 5x1 10 10 0.1 7.5
10X Buffer G BG5 5x1 10 10 50 0.1 7.5
10X Buffer O BO5 5x1 50 10 100 0.1 7.5
10X Buffer R BR5 5x1 10 10 100 0.1 8.5
10X Buffer Tango™ BY5 5x1 33 10 66 0.1 7.9
Buffer Set for Restriction Enzymes B30
1 ml of each buffer
Unique buffers
10X Buffer AarI, AjiI, Bpu10I, ScaI, PasI B27 1 10 10 100 0.1 6.5
10X Buffer BamHI, Lsp1109I, SgeI B57 5x1 10 5 100 0.02 0.1 8.0
10X Buffer BfuI B59 1 50 15 100 0.1 7.9
10X Buffer BseXI B31 1 50 2 100 0.1 7.5
10X Buffer Bsp143I B13 1 33 10 66 0.02 0.1 7.9
10X Buffer Cfr9I B02 1 10 5 200 0.1 7.2
10X Buffer Cfr10I B04 1 10 5 100 0.02 0.1 8.0
10X Buffer Eam1105I B25 1 10 5 100 0.1 7.5
10X Buffer Ecl136II, PacI,SacI B26 1 10 10 0.1 6.5
10X Buffer Eco52I B22 1 10 3 100 0.1 8.5
10X Buffer EcoRI B12 5x1 50 10 100 0.02 0.1 7.5
10X Buffer KpnI B29 1 10 10 0.02 0.1 7.5
10X Buffer SdaI B24 1 37 15 150 0.1 7.0
10X Buffer SduI, Ppu21I B23 1 10 3 150 0.1 7.2
10X Buffer TaqI B28 1 10 5 100 0.1 8.0
Dilution Buffer for Restriction Enzymes B19 5x1 ml 10 100 1 1 50 0.2 7.4
163
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Recommended Reaction Conditions
Table 1.6. Reaction conditions for restriction enzymes.
Enzyme
Units for overnight
incubation, u/µg DNA
Thermal inactivation
in 20 min
Reco- mmended buffer for
100% activity
Enzyme activity in Fermentas buffers, %Tango™ buffer
for double digestion
B (blue) 1X
G (green) 1X
O (orange) 1X
R (red) 1X
Tango™ (yellow)1X 2X
AanI (PsiI) 0.5 65° C Tango™ 50-100 50-100 0-20 0-20 100 20-50 1X or 2X
AarI 1.0 65° CUnique (+oligo)
NR NR0-20
(+oligo)0-20
(+oligo)NR
50-100 (+oligo)
2X (+oligo)
AasI (DrdI) 0.1 80° C B 100 20-50 0-20 0-20 50-100* 0-20 1X*
AatII 0.3 65° C Tango™ 50-100 20-50 0-20 0-20 100 20-50 1X or 2X
Acc65I (Asp718I) 0.3 65° C O 0-20 20-50 100 20-50 20-50 50-100 1X or 2X
AdeI (DraIII ) 0.5 NO G 0-20 100 20-50 100 100* 20-50 1X* or 2X
AjiI (BmgBI) 0.5 65° C Unique NR NR 20-50* NR NR 20-50* 2X*
AjuI 0.5 65° CR
(+SAM)0-20
(+SAM)50-100 (+SAM)
20-50 (+SAM)
100 (+SAM)
50-100 (+SAM)
50-100 (+SAM)
1X or 2X (+SAM)
AlfI 1.0 65° CR
(+SAM)0-20
(+SAM)0-20
(+SAM)0-20
(+SAM)100
(+SAM)0-20
(+SAM)20-50
(+SAM)2X
(+SAM)
AloI (30°C) 0.1 65° C R 0-20 0-20 0-20 100 20-50 100 1X or 2X
AluI 0.1 65° C Tango™ 50-100 0-20 0-20 0-20 100 20-50 1X or 2X
Alw21I (BsiHKAI) 0.1 65° C O 0-20 20-50 100 50-100 20-50 50-100 1X or 2X
Alw26I (BsmAI) 0.2 65° C Tango™ 50-100 100 0-20 0-20 100 100 1X or 2X
Alw44I (ApaLI) 0.1 65° C Tango™ 50-100 100 0-20 50-100 100 50-100 1X or 2X
ApaI 0.2 65° C B 100 20-50 0-20 0-20 20-50 0-20 1X
BamHI 0.5 80° C (10 u) Unique 20-50* 100 20-50 50-100* 100* 50-100 1X* or 2X
BauI (BssSI) 0.2 65° C Tango™ 0-20 50-100 0-20 50-100 100 50-100 1X or 2X
BclI (55°C) 0.1 80° C (10 u) G 20-50 100 20-50 20-50 100* 100 1X* or 2X
BcnI (NciI) 0.2 65° C Tango™ 20-50 50-100 50-100 50-100 100 50-100 1X or 2X
BcuI (SpeI) 0.5 NO Tango™ 50-100 50-100 0-20 20-50 100 0-20 1X
BdaI (30°C) 1.0 65° CG
(+SAM)NR
100 (+SAM)
0-20 (+SAM)
20-50 (+SAM)
50-100*(+SAM)
50-100 (+SAM)
1X* or 2X(+SAM)
BfiI (BmrI) 1.0 65° C Tango™ 20-50 20-50 0-20 0-20 100 0-20 1X
BfmI (SfcI) 1.0 65° C Tango™ 0-20 50-100 0-20 0-20 100 20-50 1X or 2X
BfuI (BciVI) 1.0 80° C Unique NR NR 0-20 0-20 NR 50-100* 2X*
BglI 0.1 65° C O 0-20 50-100 100 100 0-20 100 2X
BglII 0.1 NO O 0-20 20-50 100 50-100 0-20 100 2X
Bme1390I (ScrFI) 0.2 80° C O 20-50 50-100 100 50-100 50-100 50-100 1X or 2X
BoxI (PshAI) 0.5 80° C Tango™ 0-20 0-20 0-20 20-50 100 20-50 1X or 2X
BpiI (BbsI) 0.3 65° C G 20-50 100 50-100 50-100 50-100 50-100 1X or 2X
BplI 0.3 65° CTango™
(+SAM)0-20
(+SAM)20-50
(+SAM)0-20
(+SAM)0-20
(+SAM)100
(+SAM)20-50
(+SAM)1X
(+SAM)
Bpu10I 0.2 80° C Unique 0-20 20-50* 50-100* 100* 50-100* 100* 1X* or 2X*
Bpu1102I (BlpI) 0.1 80° C Tango™ 50-100 50-100 20-50 20-50 100 20-50 1X or 2X
BseDI (BsaJI) (55°C) 0.2 80° C Tango™ 50-100 20-50 0-20 0-20 100 50-100 1X or 2X
BseGI (BtsCI) (55°C) 0.2 80° C Tango™ 20-50 50-100 20-50 20-50 100 20-50 1X or 2X
BseJI (BsaBI) (65°C) 0.1 NO O NR 100* 100 NR NR 100* 2X*
BseLI (BslI) (55°C) 0.1 NO Tango™ 20-50 100 50-100 20-50 100 50-100 1X or 2X
BseMI (BsrDI) (55°C) 0.3 80° C R 0-20 20-50 0-20 100 50-100 50-100 1X or 2X
BseMII (BspCNI) (55°C)
0.5 80° CTango™
(+SAM)50-100 (+SAM)
50-100 (+SAM)
50-100 (+SAM)
50-100 (+SAM)
100 (+SAM)
50-100 (+SAM)
1X or 2X (+SAM)
BseNI (BsrI) (65°C) 0.1 80° C B 100 20-50 0-20 0-20 50-100 20-50 1X or 2X
BseSI (Bme1580I) (55°C) 0.1 80° C (10 u) G 20-50 100 0-20 20-50 50-100 0-20 1X
BseXI (BbvI) (65°C) 0.3 80° C Unique NR NR NR NR NR NR NR
Bsh1236I (BstUI) 0.1 65°C R 0-20 0-20 50-100 100 20-50 50-100 1X or 2X
Bsh1285I (BsiEI) 0.2 80° C G 20-50 100 20-50 0-20 0-20 20-50 2X
(continued on next page)
164
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
(continued on next page)
Enzyme
Units for overnight
incubation, u/µg DNA
Thermal inactivation
in 20 min
Reco- mmended buffer for
100% activity
Enzyme activity in Fermentas buffers, %Tango™ buffer
for double digestion
B (blue) 1X
G (green) 1X
O (orange) 1X
R (red) 1X
Tango™ (yellow)1X 2X
BshNI (BanI) 0.3 65° C O 0-20 20-50 100 50-100 0-20 100 2X
BshTI (AgeI) 0.1 80° C O 0-20 20-50 100 50-100 20-50 20-50 1X or 2X
Bsp68I (NruI) 0.2 65° C O 0-20 20-50 100 50-100 20-50 50-100 1X or 2X
Bsp119I (BstBI) 0.1 80° C Tango™ 20-50 0-20 0-20 0-20 100 100 1X or 2X
Bsp120I (PspOMI) 0.1 80° C B 100 20-50 0-20 20-50 50-100 0-20 1X
Bsp143I (Sau3AI) 0.1 65° C Unique 20-50 20-50 0-20 0-20 50-100 20-50 1X or 2X
Bsp1407I (BsrGI) 0.1 65° C Tango™ 0-20 20-50 0-20 20-50 100 50-100 1X or 2X
BspLI (NlaIV) 0.3 65° C Tango™ 50-100 50-100 0-20 20-50 100 20-50 1X or 2X
BspOI (BmtI) 0.2 80° C O 0-20 0-20 100 100 0-20 20-50 2X
BspPI (AlwI) (55°C) 0.5 80° C Tango™ 20-50 20-50 0-20 0-20 100 0-20 1X
BspTI (AfIII ) 0.1 65° C O 0-20 0-20 100 20-50 0-20 50-100 2X
Bst1107I (BstZ17I) 0.1 80° C O 20-50 50-100 100 100 20-50 100 1X or 2X
BstXI (55°C) 0.1 80° C O 20-50 100 100 50-100 50-100 100 1X or 2X
Bsu15I (ClaI) 0.1 65° C Tango™ 20-50 20-50 20-50 20-50 100 20-50 1X or 2X
BsuRI (HaeIII) 0.1 80° C R 20-50 20-50 50-100 100 50-100 100 1X or 2X
BveI (BspMI) 0.2 65° CO
(+oligo)0-20
(+oligo)20-50
(+oligo)100
(+oligo)20-50
(+oligo)50-100 (+oligo)
100 (+oligo)
1X or 2X (+oligo)
CaiI (AlwNI) 0.2 65° C Tango™ 20-50 20-50 20-50 50-100 100 50-100 1X or 2X
CfrI (EaeI) 1.0 65° C Tango™ 50-100* 50-100 0-20 0-20 100 0-20 1X
Cfr9I (XmaI) 0.2 65° C Unique 0-20 0-20 0-20 0-20 20-50 0-20 1X
Cfr10I (BsrFI) 0.1 NO Unique 0-20 20-50 20-50 50-100* 20-50 50-100 1X or 2X
Cfr13I (Sau96I) 0.3 65° C Tango™ 50-100 50-100 20-50 20-50 100 20-50 1X or 2X
Cfr42I (SacII) 0.1 65° C B 100 50-100 0-20 0-20 50-100 0-20 1X
CpoI (RsrII) 0.5 65° C Tango™ 20-50 50-100 50-100 20-50 100 50-100 1X or 2X
CseI (HgaI) 0.5 80° C (10 u) R NR 50-100* 50-100 100 100* 50-100 1X* or 2X
Csp6I (CviQI) 0.1 65° C B 100 50-100 0-20 0-20 50-100 0-20 1X
DpnI 0.1 80° C Tango™ 100 100 50-100 50-100 100 50-100 1X or 2X
DraI 0.1 65° C Tango™ 50-100 50-100 20-50 20-50 100 50-100 1X or 2X
Eam1104I (EarI) 0.5 65° C Tango™ 50-100 50-100 0-20 0-20 100 0-20 1X
Eam1105I (AhdI) 0.1 65° C Unique 20-50 50-100 0-20 0-20 50-100 20-50 1X or 2X
Ecl136II (EcoICRI) 0.2 65° C Unique 50-100 20-50 0-20 0-20 50-100 0-20 1X
Eco24I (BanII) 0.2 65° C Tango™ 50-100 50-100 0-20 20-50 100 0-20 1X
Eco31I (BsaI) 0.3 65° C G 50-100 100 0-20 0-20 50-100 20-50 1X or 2X
Eco32I (EcoRV) 0.1 80° C R 0-20 50-100 50-100 100 20-50 100 1X or 2X
Eco47I (AvaII) 0.3 65° C R 0-20 50-100 50-100 100 50-100 50-100 1X or 2X
Eco47III (AfeI) 0.1 65° C O 0-20 20-50 100 100 50-100 100 1X or 2X
Eco52I (EagI) 0.2 65° C Unique 0-20 0-20 0-20 20-50 0-20 20-50 2X
Eco57I (AcuI) 1.0 65° CG
(+SAM)100
(+SAM)100
(+SAM)20-50
(+SAM)20-50
(+SAM)50-100 (+SAM)
50-100 (+SAM)
1X or 2X (+SAM)
Eco57MI 1.0 65° CB
(+SAM)100
(+SAM)50-100 (+SAM)
0-20 (+SAM)
20-50 (+SAM)
50-100 (+SAM)
0-20 (+SAM)
1X (+SAM)
Eco72I (PmlI) 0.5 65° C Tango™ NR NR 0-20 0-20 100 20-50 1X or 2X
Eco81I (Bsu36I) 0.1 80° C Tango™ 50-100 100 0-20 0-20 100 0-20 1X
Eco88I (AvaI) 0.2 65° C Tango™ 100 50-100 0-20 0-20 100 20-50 1X or 2X
Eco91I (BstEII) 0.1 65° C O 20-50 20-50 100 50-100 50-100 100 1X or 2X
Eco105I (SnaBI) 0.5 65° C Tango™ 100* 50-100 0-20 0-20 100 0-20 1X
Eco130I (StyI) 0.2 65° C O 0-20 20-50 100 50-100 50-100 100 1X or 2X
Eco147I (StuI) 0.1 80° C B 100 50-100 20-50 20-50 50-100 0-20 1X
EcoO109I (DraII) 0.2 65° C Tango™ 50-100 20-50 20-50 20-50 100 100 1X or 2X
EcoRI 0.2 65° C Unique 0-20 NR 100 100* NR 100 2X
Table 1.6. Reaction conditions for restriction enzymes.
165
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
(continued on next page)
Enzyme
Units for overnight
incubation, u/µg DNA
Thermal inactivation
in 20 min
Reco- mmended buffer for
100% activity
Enzyme activity in Fermentas buffers, %Tango™ buffer
for double digestion
B (blue) 1X
G (green) 1X
O (orange) 1X
R (red) 1X
Tango™ (yellow)1X 2X
EcoRII 0.1 80° C O 20-50 50-100 100 50-100 20-50 50-100 1X or 2X
EheI (SfoI) 1.0 65° C Tango™ 20-50 50-100 0-20 0-20 100 20-50 1X or 2X
Esp3I (BsmBI) 0.2 65° CTango™
(+DTT)100
(+DTT)20-50 (+DTT)
0-20 (+DTT)
0-20 (+DTT)
100 (+DTT)
0-20 (+DTT)
1X (+DTT)
FaqI (BsmFI) 1.0 80° CTango™
(+SAM)20-50
(+SAM)20-50
(+SAM)0-20
(+SAM)0-20
(+SAM)100
(+SAM)20-50
(+SAM)1X or 2X (+SAM)
FspAI 0.2 65° C O 0-20 0-20 100 50-100 0-20 50-100 2X
FspBI (BfaI) 0.3 65° C Tango™ 50-100 20-50 0-20 0-20 100 0-20 1X
GsuI (BpmI) (30°C) 1.0 65° C B 100 50-100 20-50 20-50 100 50-100 1X or 2X
HhaI 0.1 NO Tango™ 50-100 50-100 20-50 20-50 100 20-50 1X or 2X
Hin1I (BsaHI) 0.1 65° C G 20-50 100 20-50 20-50 20-50 20-50 1X or 2X
Hin1II (NlaIII ) 0.3 65° C G 50-100 100 20-50 50-100 50-100 50-100 1X or 2X
Hin4I 0.2 65° CTango™ (+SAM)
20-50 (+SAM)
20-50 (+SAM)
0-20 (+SAM)
0-20 (+SAM)
100 (+SAM)
0-20 (+SAM)
1X (+SAM)
Hin6I (HinP1I) 0.1 65° C Tango™ 50-100 50-100 20-50 20-50 100 50-100 1X or 2X
HincII (HindII) 0.1 65° C Tango™ 50-100 50-100 20-50 50-100 100 50-100 1X or 2X
HindIII 0.1 80° C R 0-20 20-50 0-20 100 50-100 50-100 1X or 2X
HinfI 0.1 65° C R 0-20 20-50 50-100 100 50-100 50-100 1X or 2X
HpaII 0.1 65° C Tango™ 50-100 50-100 0-20 20-50 100 20-50 1X or 2X
HphI 0.1 65° C B 100 0-20 0-20 0-20 20-50 0-20 1X
Hpy8I (MjaIV) 0.1 80° C Tango™ 50-100 50-100 0-20 20-50 100 50-100 1X or 2X
HpyF3I (DdeI) 0.2 65° C Tango™ 20-50 20-50 20-50 20-50 100 50-100 1X or 2X
HpyF10VI (MwoI) 0.1 80° C Tango™ 0-20 0-20 0-20 0-20 100 50-100 1X or 2X
KpnI 0.2 80° C Unique 20-50 0-20 0-20 0-20 20-50 0-20 1X
Kpn2I (BspEI) (55°C) 0.2 80° C Tango™ 50-100 50-100 0-20 20-50 100 50-100 1X or 2X
KspAI (HpaI) 0.5 65° C B 100 50-100* 20-50 20-50 100* 50-100 1X* or 2X
LguI (SapI) 0.5 65° C Tango™ 20-50 50-100 20-50 20-50 100 20-50 1X or 2X
Lsp1109I (BbvI) 0.5 65° C Unique 0-20 20-50* 50-100* 100* 20-50* 20-50* 1X* or 2X*
LweI (SfaNI) 0.2 65° C Tango™ 0-20 0-20 0-20 20-50 100 20-50 1X or 2X
MauBI 0.1 65° C Tango™ 0-20 0-20 0-20 0-20 100 0-20 1X
MbiI (BsrBI) 0.2 65° C Tango™ 20-50 100 20-50 20-50 100 20-50 1X or 2X
MboI 0.1 65° C R 50-100 50-100 50-100 100 50-100 100 1X or 2X
MboII 1.0 65° C B 100 50-100 20-50 0-20 50-100 20-50 1X or 2X
MlsI (MscI) 0.5 65° C R 0-20 20-50 0-20 100 20-50 50-100 1X or 2X
MluI 0.1 80° C R 0-20 20-50 50-100 100 20-50 50-100 1X or 2X
MnlI 0.5 65° C G 50-100 100 20-50 20-50 20-50 20-50 1X or 2X
Mph1103I (NsiI) 0.3 65° C R 0-20 50-100 20-50 100 50-100 50-100 1X or 2X
MreI (Sse232I) 0.5 80° C G 20-50 100 0-20 0-20 50-100 0-20 1X
MspI (HpaII) 0.3 80° C Tango™ 50-100 50-100 0-20 0-20 100 50-100 1X or 2X
MssI (PmeI) 0.5 65° C B 100 0-20 0-20 0-20 20-50 0-20 1X
MunI (MfeI) 0.1 65° C G 100 100 0-20 0-20 100 0-20 1X
MvaI (BstNI) 0.1 NO R 20-50 20-50 50-100 100 20-50* 100 1X* or 2X
Mva1269I (BsmI) 0.1 65° C R 0-20 20-50 50-100 100 0-20 50-100 2X
NcoI 0.1 65° C Tango™ 20-50 20-50 20-50 50-100 100 100 1X or 2X
NdeI 0.2 65° C O 0-20 0-20 100 50-100 0-20 50-100 2X
NheI 0.2 65° C Tango™ 100 20-50 0-20 0-20 100 0-20 1X
NmuCI (Tsp45I) 0.1 65° C R 0-20 20-50 50-100 100 20-50 50-100 1X or 2X
NotI 0.1 80° C O 0-20 0-20 100 20-50 0-20 20-50 2X
NsbI (FspI) 0.1 65° C Tango™ 20-50 50-100 0-20 20-50 100 20-50 1X or 2X
OliI (AleI) 0.2 65° C R 0-20 0-20 0-20 100 0-20 50-100 2X
Table 1.6. Reaction conditions for restriction enzymes.
166
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
(continued on next page)
Enzyme
Units for overnight
incubation, u/µg DNA
Thermal inactivation
in 20 min
Reco- mmended buffer for
100% activity
Enzyme activity in Fermentas buffers, %Tango™ buffer
for double digestion
B (blue) 1X
G (green) 1X
O (orange) 1X
R (red) 1X
Tango™ (yellow)1X 2X
PacI 0.3 65° C Unique 20-50 20-50 0-20 0-20 0-20 0-20 NR
PaeI (SphI) 0.1 65° C B 100 50-100 0-20 0-20 50-100 0-20 1X
PagI (BspHI) 0.2 80° C O 0-20 50-100 100 NR NR NR NR
PasI (55°C) 0.2 80° C Unique NR NR NR NR NR NR NR
PauI (BssHII) 0.2 80° C R 0-20 0-20 100 100 0-20 100 2X
PdiI (NaeI) 0.5 65°C Tango™ 50-100 20-50 0-20 0-20 100 50-100 1X or 2X
PdmI (XmnI) 0.5 65° C Tango™ 20-50 50-100 0-20 0-20 100 0-20 1X
PfeI (TfiI) 0.1 65° C O 0-20 20-50 100 50-100 20-50 50-100 1X or 2X
Pfl23II (BsiWI) 0.3 65° C Tango™ 20-50 50-100 20-50 20-50 100 0-20 1X
PfoI 0.1 65° C Tango™ 0-20 20-50 50-100 0-20 100 50-100 1X or 2X
PpiI (30°C) 0.1 65° C R 0-20 0-20 0-20 100 50-100 50-100 1X or 2X
Ppu21I (BsaAI) (30°C) 0.5 65° C Unique 50-100* 100* 20-50 NR NR NR NR
PscI (PciI) 0.2 65° C Tango™ 20-50 20-50 0-20 0-20 100 0-20 1X
Psp5II (PpuMI) 0.2 80° C G 0-20 100 20-50 20-50 50-100 100 1X or 2X
Psp1406I (AclI) 0.5 65° C Tango™ 100 50-100 0-20 20-50 100 0-20 1X
PstI 0.2 NO O 50-100 50-100 100 100 50-100 50-100 1X or 2X
PsuI (BstYI) 0.5 80° C B 100 20-50 0-20 0-20 50-100 0-20 1X
PsyI (Tth111I) 0.1 80° C B 100 50-100 0-20 0-20 50-100 0-20 1X
PvuI 0.2 80° C (10 u) R 0-20 20-50 50-100 100 50-100 100 1X or 2X
PvuII 0.2 NO G 50-100* 100 20-50 50-100 20-50* 20-50* 1X* or 2X*
RsaI 0.2 80° C Tango™ 50-100 20-50 0-20 0-20 100 0-20 1X
RseI (MslI) 0.5 65° C R 0-20 50-100 50-100 100 20-50 100 1X or 2X
SacI 0.2 65° C Unique 50-100 20-50 0-20 0-20 50-100 20-50 1X or 2X
SalI 0.1 65° C O 0-20 0-20 100 20-50 0-20 50-100 2X
SatI (Fnu4HI) 0.1 65° C G 20-50 100 20-50 20-50 50-100 20-50 1X or 2X
ScaI 0.5 80° C (10 u) Unique 0-20 0-20 0-20 0-20 0-20 0-20 NR
SchI (MlyI) 0.2 65° C Tango™ 20-50 50-100 0-20 0-20 100 0-20 1X
SdaI (SbfI) 0.3 80° C Unique NR NR 0-20 0-20 NR 20-50 2X
SduI (Bsp1286I) 0.3 65° C Unique NR 50-100* 50-100 0-20 NR NR NR
SfaAI (AsiSI) 0.2 80° C Tango™ 50-100 0-20 0-20 0-20 100 0-20 1X
SfiI (50°C) 0.2 NO G 50-100 100 20-50 0-20 100 0-20 1X
SgeI NR 65° C Unique 0-20 0-20 0-20 NR NR NR NR
SgrDI 0.3 65° C R 0-20 0-20 0-20 100 NR 100 2X
SgsI (AscI) 0.1 65° C Tango™ 0-20 0-20 0-20 50-100 100 50-100 1X or 2X
SmaI (30°C) 0.2 65° C Tango™ 50-100 0-20 0-20 0-20 100 0-20 1X
SmiI (SwaI) (30°C) 0.1 65° C O 0-20 0-20 100 20-50 0-20 20-50 2X
SmoI (SmlI) (55°C) 0.2 80° C Tango™ 50-100 20-50 0-20 20-50 100 20-50 1X or 2X
SmuI (FauI) 0.2 65° C Tango™ 50-100 50-100 0-20 20-50 100 20-50 1X or 2X
SsiI (AciI) 0.5 65° C O NR 20-50 100 50-100 NR 100 2X
SspI 0.1 65° C G 20-50 100 0-20 50-100 100 20-50 1X or 2X
SspDI (KasI) 0.1 80° C Tango™ 20-50 0-20 NR 0-20 100 20-50 1X or 2X
TaaI (HpyCH4III) (65°C) 0.2 NO Tango™ 0-20 0-20 0-20 50-100 100 100 1X or 2X
TaiI (MaeII) (65°C) 0.3 NO R 50-100 50-100 20-50 100 100 50-100 1X or 2X
TaqI (65°C) 0.3 NO Unique 0-20 20-50 20-50 20-50 20-50 20-50 1X or 2X
TasI (Tsp509I) (65°C) 0.3 NO B 100 50-100 20-50 0-20 20-50 0-20 1X
TatI (65°C) 0.2 NO Tango™ NR 50-100* 20-50 20-50 100* 0-20 1X*
TauI (55°C) 1.0 NO B 100 50-100 0-20 0-20 20-50 0-20 1X
Tru1I (MseI) (65°C) 0.2 NO R 50-100 50-100 20-50 100 50-100 100 1X or 2X
TscAI (TspRI) (65°C) 0.2 NO Tango™ 50-100 50-100 20-50 20-50 100 20-50 1X or 2X
Table 1.6. Reaction conditions for restriction enzymes.
167
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Enzyme
Units for overnight
incubation, u/µg DNA
Thermal inactivation
in 20 min
Reco- mmended buffer for
100% activity
Enzyme activity in Fermentas buffers, %Tango™ buffer
for double digestion
B (blue) 1X
G (green) 1X
O (orange) 1X
R (red) 1X
Tango™ (yellow)1X 2X
TsoI (55°C) 1.0 80° CG
(+SAM)NR
(+SAM)100
(+SAM)50-100 (+SAM)
0-20 (+SAM)
50-100 (+SAM)
20-50 (+SAM)
1X or 2X (+SAM)
TstI 0.1 NO R 0-20 0-20 0-20 100 0-20 100 2X
Van91I (PflMI) 0.1 65° C R 0-20 50-100 50-100 100 20-50 50-100 1X or 2X
VspI (AseI) 0.1 65° C O 0-20 50-100 100 20-50 100 100 1X or 2X
XagI (EcoNI) 0.1 65° C R 0-20 20-50 50-100 100 20-50 50-100 1X or 2X
XapI (ApoI) 0.1 80° C Tango™ 50-100 100 0-20 0-20 100 20-50 1X or 2X
XbaI 0.1 65°C Tango™ 50-100 50-100 20-50 0-20 100 50-100 1X or 2X
XceI (NspI) 0.2 65° C Tango™ 50-100 0-20 0-20 0-20 100 0-20 1X
XhoI 0.1 80° C R 0-20 50-100 50-100 100 20-50 100 1X or 2X
XmaJI (AvrII) 0.2 80° C Tango™ 20-50 50-100 50-100 50-100 100 50-100 1X or 2X
XmiI (AccI) 0.1 65°C B 100 0-20 0-20 0-20 50-100 20-50 1X or 2X
Nicking enzymes
Nb.Bpu10I 0.3 80° C R 0-20 20-50 20-50 100 20-50 50-100 1X or 2X
Nt.Bpu10I 0.3 65° C R 0-20 0-20 100 100 0-20 50-100 2X
Nb.Mva1269I 0.3 80° C O 0-20 20-50 100 100 20-50 50-100 1X or 2X
Homing enzyme
I-SceI 0.5 65° C Tango™ 50-100 50-100 50-100 50-100 100 50-100 1X or 2X
Table 1.6. Reaction conditions for restriction enzymes.
* – star activity appears at a greater than 5-fold overdigestion (5 units x 1 hour). NR – buffer is not recommended, because of high star activity.NO – no thermal inactivation.80°C (10 u) – only small amounts of the restriction enzyme (up to 10 units) can be inactivated at 80°C in 20 min.
EnzymeOptimal
temperatureActivity at 37°C, %
AloI 30 20BclI 55 50BdaI 30 30BseDI (BsaJI) 55 10BseGI (BtsCI) 55 25BseJI (BsaBI) 65 <10BseLI (BslI) 55 40BseMI (BsrDI) 55 20BseMII (BspCNI) 55 30BseNI (BsrI) 65 <10BseSI (Bme1580I) 55 20BseXI (BbvI) 65 10BspPI (AlwI) 55 30BstXI 55 50GsuI (BpmI) 30 70Kpn2I (BspEI) 55 50PasI 55 30
EnzymeOptimal
temperatureActivity at 37°C, %
PpiI 30 60Ppu21I (BsaAI) 30 <30SfiI 50 10SmaI* 30 50SmiI (SwaI) 30 70SmoI (SmlI) 55 10TaaI (HpyCH4III) 65 10TaiI (MaeII) 65 <10TasI (Tsp509I) 65 <10TaqI* 65 10TauI 55 30TatI 65 20Tru1I (MseI)* 65 10TscAI (TspRI) 65 <5TsoI 55 10
* – high concentration enzyme preparations are available for incubation at non-optimal temperature.
Activity of Mesophilic and Thermophilic Enzymes at 37°CTable 1.7. Activity of mesophilic and thermophilic enzymes at 37°C.
168
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Double Digestion in Universal Tango™ Buffer
Table 1.8. Double digestion in universal Tango™ buffer.
Fermentas enzyme
Buffers for double digestionsRecommended buffer
Tango™
1X 2XAanI (PsiI) Tango™
AarI AarI +oligo NR +oligo
AasI (DrdI) B * NRAatII Tango™
Acc65I (Asp718I) OAdeI (DraIII ) G *AjiI (BmgBI) AjiI NR *AjuI R +SAM +SAM +SAM
AlfI R +SAM NR +SAM
AloI (30°C) RAluI Tango™
Alw21I (BsiHKAI) OAlw26I (BsmAI) Tango™
Alw44I (ApaLI) Tango™
ApaI B NRBamHI BamHI *BauI (BssSI) Tango™
BclI (55°C) G *BcnI (NciI) Tango™
BcuI (SpeI) Tango™ NRBdaI (30°C) G +SAM +SAM * +SAM
BfiI (BmrI) Tango™ NRBfmI (SfcI) Tango™
BfuI (BciVI) BfuI NR *BglI O NRBglII O NRBme1390I (ScrFI) OBoxI (PshAI) Tango™
BpiI (BbsI) GBplI Tango™+SAM +SAM NRBpu10I Bpu10I * *Bpu1102I (BlpI) Tango™
BseDI (BsaJI) (55°C) Tango™
BseGI (BtsCI) (55°C) Tango™
BseJI (BsaBI) (65°C) O NR *BseLI (BslI) (55°C) Tango™
BseMI (BsrDI) (55°C) RBseMII (BspCNI) (55°C) Tango™+SAM +SAM +SAM
BseNI (BsrI) (65°C) BBseSI (Bme1580I) (55°C) G NRBseXI (BbvI) (65°C) BseXI NR NRBsh1236I (BstUI) RBsh1285I (BsiEI) G NRBshNI (BanI) O NRBshTI (AgeI) OBsp68I (NruI) OBsp119I (BstBI) Tango™
Bsp120I (PspOMI) B NRBsp143I (Sau3AI) Bsp143IBsp1407I (BsrGI) Tango™
BspLI (NlaIV) Tango™
BspOI (BmtI) O NRBspPI (AlwI) (55°C) Tango™ NRBspTI (AflII) O NR
Fermentas enzyme
Buffers for double digestionsRecommended buffer
Tango™
1X 2XBst1107I (BstZ17I) OBstXI (55°C) OBsu15I (ClaI) Tango™
BsuRI (HaeIII) RBveI (BspMI) O +oligo +oligo +oligo
CaiI (AlwNI) Tango™
CfrI (EaeI) Tango™ NRCfr9I (XmaI) Cfr9I NRCfr10I (BsrFI) Cfr10ICfr13I (Sau96I) Tango™
Cfr42I (SacII) B NRCpoI (RsrII) Tango™
CseI (HgaI) R *Csp6I (CviQI) B NRDpnI Tango™
DraI Tango™
Eam1104I (EarI) Tango™ NREam1105I (AhdI) Eam1105IEcl136II (EcoICRI) Ecl136II NREco24I (BanII) Tango™ NREco31I (BsaI) GEco32I (EcoRV) REco47I (AvaII) REco47III (AfeI) OEco52I (EagI) Eco52I NREco57I (AcuI) G +SAM +SAM +SAM
Eco57MI B +SAM +SAM NREco72I (PmII) Tango™
Eco81I (Bsu36I) Tango™ NREco88I (AvaI) Tango™
Eco91I (BstEII) OEco105I (SnaBI) Tango™ NREco130I (StyI) OEco147I (StuI) B NREcoO109I (DraII) Tango™
EcoRI EcoRI NREcoRII OEheI (SfoI) Tango™
Esp3I (BsmBI) Tango™+DTT +DTT NRFaqI (BsmFI) Tango™+SAM +SAM +SAM
FspAI O NRFspBI (BfaI) Tango™ NRGsuI (BpmI) (30°C) BHhaI Tango™
Hin1I (BsaHI) GHin1II (NlaIII ) GHin4I Tango™+SAM +SAM NRHin6I (HinP1I) Tango™
HincII (HindII) Tango™
HindIII RHinfI RHpaII Tango™
HphI B NRHpy8I (MjaIV) Tango™
(continued on next page)
169
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.8. Double digestion in universal Tango™ buffer.
Fermentas enzyme
Buffers for double digestionsRecommended buffer
Tango™
1X 2XHpyF3I (DdeI) Tango™
HpyF10VI (MwoI) Tango™
KpnI KpnI NRKpn2I (BspEI) (55°C) Tango™
KspAI (HpaI) B *LguI (SapI) Tango™
Lsp1109I (BbvI) Lsp1109I * *LweI (SfaNI) Tango™
MauBI Tango™ NRMbiI (BsrBI) Tango™
MboI RMboII BMlsI (MscI) RMluI RMnlI GMph1103I (NsiI) RMreI (Sse232I) G NRMspI (HpaII) Tango™
MssI (PmeI) B NRMunI (MfeI) G NRMvaI (BstNI) R *Mva1269I (BsmI) R NRNcoI Tango™
NdeI O NRNheI Tango™ NRNmuCI (Tsp45I) RNotI O NRNsbI (FspI) Tango™
OliI (AleI) R NRPaeI (SphI) B NRPacI PacI NR NRPagI (BspHI) O NR NRPasI (55°C) PasI NR NRPauI (BssHII) R NRPdiI (NaeI) Tango™
PdmI (XmnI) Tango™ NRPfeI (TfiI) OPfl23II (BsiWI) Tango™ NRPfoI Tango™
PpiI (30°C) RPpu21I (BsaAI) (30°C) Ppu21I NR NRPscI (PciI) Tango™ NRPsp5II (PpuMI) GPsp1406I (AciI) Tango™ NRPstI OPsuI (BstYI) B NRPsyI (Tth111I) B NRPvuI RPvuII G * *RsaI Tango™ NRRseI (MslI) RSacI SacISalI O NRSatI (Fnu4HI) GScaI ScaI NR NR
Fermentas enzyme
Buffers for double digestionsRecommended buffer
Tango™
1X 2XSchI (MlyI) Tango™ NRSdaI (SbfI) SdaI NRSduI (Bsp1286I) SduI NR NRSfaAI (AsiSI) Tango™ NRSfiI (50°C) G NRSgeI SgeI NR NRSgrDI R NRSgsI (AscI) Tango™
SmaI (30°C) Tango™ NRSmiI (SwaI) (30°C) O NRSmoI (SmoI) (55°C) Tango™
SmuI (FauI) Tango™
SsiI (AciI) O NRSspI GSspDI (KasI) Tango™
TaaI (HpyCH4III) (65°C) Tango™
TaiI (MaeII) (65°C) RTaqI (65°C) TaqITasI (Tsp509I) (65°C) B NRTauI (55°C) B NRTatI (65°C) Tango™ * NRTru1I (MseI) (65°C) RTscAI (TspRI) (65°C) Tango™
TsoI (55°C) G +SAM +SAM +SAM
TstI R NRVan91I (PflMI) RVspI (AseI) OXagI (EcoNI) RXapI (ApoI) Tango™
XbaI Tango™
XceI (NspI) Tango™ NRXhoI RXmaJI (AvrII) Tango™
XmiI (AccI) BI-SceI Tango™
Nb.Bpu10I RNt.Bpu10I R NRNb.Mva1269I O
* – star activity appears at a greater than 5-fold overdigestion (5 units x 1 hour).
Optimal temperature for the enzymes listed is 37°C unless otherwise indicated in parenthesis.
NR – buffer is not recommended since enzyme activity is less than 20% or star activity is too high.
Cleavage efficiency20-50%50-100%
170
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Fermentas enzyme
Units of enzyme, needed for DNA cleavage in 16 h
AanI (PsiI) 5AarI 5AasI (DrdI) 5AatII 5Acc65I (Asp718I) 5AdeI (DraIII ) 10AjiI (BmgBI) 5AjuI 5
AlfI 5Alw44I (ApaLI) 5ApaI 5BamHI 5BauI (BssSI) 5BcuI (SpeI) 10BfuI (BciVI) 5BglI 5BglII 5BoxI (PshAI) 10BpiI (BbsI) 5BplI 10Bpu1102I (BlpI) 5BseSI (Bme1580I) 5Bsh1285I (BsiEI) 5BshNI (BanI) 5BshTI (AgeI) 20Bsp68I (NruI) 5Bsp119I (BstBI) 5Bsp120I (PspOMI) 5Bsp1407I (BsrGI) 10BspOI (BmtI) 20BspTI (AflII) 5Bst1107I (BstZ17I) 5BstXI 5Bsu15I (ClaI) 5CaiI (AlwNI) 5CfrI (EaeI) 5Cfr9I (XmaI) 5Cfr10I (BsrFI) 5Cfr42I (SacII) 20DraI 5Eam1104I (EarI) 5Eam1105I (AhdI) 5Ecl136II (EcoICRI) 5Eco24I (BanII) 5Eco31I (BsaI) 5Eco32I (EcoRV) 5Eco47III (AfeI) 5Eco52I (EagI) 5Eco72I (PmlI) 5Eco81I (Bsu36I) 5Eco88I (AvaI) 5Eco91I (BstEII) 5Eco105I (SnaBI) 5Eco130I (StyI) 5Eco147I (StuI) 5
Fermentas enzyme
Units of enzyme, needed for DNA cleavage in 16 h
EcoO109I (DraII) 5EcoRI 5EheI (SfoI) 20Esp3I (BsmBI) 5FspBI (BfaI) 10Hin1I (BsaHI) 5HindIII 5KpnI 5Kpn2I (BspEI) (55°C) 5KspAI (HpaI) 5LguI (SapI) 5MauBI 5MbiI (BsrBI) 5MlsI (MscI) 20MluI 5MssI (PmeI) 5MunI (MfeI) 5Mva1269I (BsmI) 5NcoI 5NdeI 5NheI 5NotI 5NsbI (FspI) 5OliI (AleI) 5PaeI (SphI) 5PacI 5PagI (BspHI) 5PasI (55°C) 10PauI (BssHII) 10PdiI (NaeI) 20PdmI (XmnI) 10Pfl23II (BsiWI) 5PfoI 10Ppu21I (BsaAI) 5PscI (PciI) 5Psp5II (PpuMI) 5Psp1406I (AcII) 10PstI 5PsyI (Tth111I) 5PvuI 5PvuII 5RseI (MslI) 5SacI 5SalI 5ScaI 20SdaI (SbfI) 5SfaAI (AsiSI) 5SfiI (50°C) 5SgeI 3SgrDI 5SgsI (AscI) 5SmaI (30°C) 5SmiI (SwaI) (30°C) 10SmoI (SmlI) (55°C) 5SspI 5
Fermentas enzyme
Units of enzyme, needed for DNA cleavage in 16 h
SspDI (KasI) 5TstI 5Van91I (PflMI) 5VspI (AseI) 5XagI (EcoNI) 5XapI (ApoI) 5XbaI 5XhoI 5XmaJI (AvrII) 10XmiI (AccI) 5I-SceI 20
Table 1.9. Digestion of agarose-embedded DNA.
Digestion of Agarose-Embedded DNA
NoteFor digestion of agarose-embedded DNA protocol see 1.11 on p.161.
171
1
1. CONVENTIONAL RESTRICTION ENZYMES
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.10. Cleavage efficiency close to the termini of PCR fragments.
Enzymebp from the recognition site to fragment end
1 2 3 4 5AanI 0 0-20 20-50AarI 20-50 50-100AasI 50-100AatII 0 0-20 20-50 50-100Acc65I 0-20 50-100AdeI 50-100AjiI 50-100AluI 0-20 20-50 50-100Alw21I 50-100Alw26I 50-100Alw44I 0 20-50 50-100ApaI 50-100BamHI 50-100BauI 0-20 20-50 50-100BcnI 20-50 50-100BclI 0 50-100BcuI 50-100BfiI 50-100BfmI 50-100BfuI 50-100BglI 20-50 50-100BglII 0 50-100Bme1390I 20-50 50-100BoxI 0 50-100BpiI 50-100Bpu10I 20-50 50-100Bpu1102I 50-100BseDI 0 50-100BseGI 50-100BseJI 0 50-100BseLI 0 50-100BseMI 0-20 50-100BseMII 50-100BseNI 0 50-100BseSI 50-100BseXI 20-50 50-100Bsh1236I 50-100Bsh1285I 0-20 50-100BshNI 50-100BshTI 20-50 50-100Bsp68I 0 50-100Bsp119I 50-100Bsp120I 20-50 50-100Bsp143I 50-100Bsp1407I 20-50 50-100BspLI 50-100BspOI 0 20-50 50-100BspPI 0 50-100BspTI 0 0-20 50-100Bst1107I 0-20 50-100BstXI 0 50-100Bsu15I 50-100BsuRI 0-20 20-50 50-100BveI 0-20 50-100CaiI 0 0-20 50-100CfrI 0 50-100Cfr9I 20-50 50-100Cfr10I 20-50 50-100Cfr13I 50-100Cfr42I 50-100CpoI 50-100CseI 50-100Csp6I 50-100DpnI 50-100DraI 0 0-20 50-100
Enzymebp from the recognition site to fragment end
1 2 3 4 5Eam1104I 0 50-100Eam1105I 0 50-100Ecl136II 50-100Eco24I 50-100Eco31I 20-50 50-100Eco32I 20-50 50-100Eco47I 50-100Eco47III 0 0-20 50-100Eco52I 0-20 50-100Eco57I 50-100Eco57MI 50-100Eco72I 0-20 50-100Eco81I 50-100Eco88I 50-100Eco91I 20-50 50-100Eco105I 20-50 50-100Eco130I 0 50-100Eco147I 0 50-100EcoO109I 50-100EcoRI 50-100EcoRII 0 0-20 20-50 50-100EheI 20-50 50-100Esp3I 50-100FaqI 0-20 50-100FspAI 0-20 20-50 50-100FspBI 20-50 50-100GsuI 50-100HhaI 50-100Hin1I 50-100Hin1II 0 0-20 50-100Hin6I 0 0-20 50-100HincII 50-100HindIII 0 0-20 50-100HinfI 50-100HpaII 0-20 50-100HphI 0 50-100Hpy8I 0-20 50-100HpyF3I 20-50 50-100HpyF10VI 50-100KpnI 50-100Kpn2I 0 50-100KspAI 20-50 50-100LguI 50-100Lsp1109I 0-20 50-100LweI 20-50 50-100MauBI 0 0-20 20-50MbiI 0 0-20 50-100MboI 50-100MboII 50-100MlsI 0-20 50-100MluI 20-50 50-100MnlI 0 50-100Mph1103I 20-50 50-100MreI 0 20-50 50-100MspI 0-20 50-100MssI 20-50 50-100MunI 20-50 50-100MvaI 0 20-50 50-100Mva1269I 0 50-100NcoI 0 50-100NdeI* 0-20 20-50 50-100NheI 0 20-50 50-100NmuCI 20-50 50-100NotI 20-50 50-100NsbI 0 0-20 50-100
Enzymebp from the recognition site to fragment end
1 2 3 4 5OliI 0-20 20-50 50-100PacI 20-50 50-100PaeI 0 0-20 20-50 50-100PagI 20-50 50-100PasI 50-100PauI 0 50-100PdiI 0-20 50-100PdmI 50-100PfeI 50-100Pfl23II 0-20 20-50 50-100PfoI 0 20-50 50-100Ppu21I 50-100PscI 0 50-100Psp5II 0 50-100Psp1406I 0-20 50-100PstI 0-20 50-100PsuI 0-20 50-100PsyI 0 50-100PvuI 20-50 50-100PvuII 50-100RsaI 50-100RseI 0 0-20 20-50 50-100SacI 50-100SalI 20-50 50-100SatI 0 50-100ScaI 0-20 50-100SchI 50-100SdaI 0-20 50-100SduI 50-100SfaAI 0 0-20 20-50SfiI 50-100SgrDI 0-20 20-50 50-100SgsI 50-100SmaI 50-100SmiI 0-20 50-100SmoI 0 50-100SmuI 50-100SsiI 50-100SspI 0-20 50-100SspDI 20-50 50-100TaaI 20-50 50-100TaiI 50-100TaqI 0 20-50 50-100TasI 0 20-50 50-100TatI 0-20 20-50 50-100TauI 20-50 50-100Tru1I 0 0-20 50-100TscAI 0 50-100TsoI 20-50 50-100Van91I 0 50-100VspI 50-100XagI 20-50 50-100XapI 20-50 50-100XbaI 20-50 50-100XceI 0 50-100XhoI 0-20 50-100XmaJI 50-100XmiI 50-100I-SceI 50-100
Some restriction enzymes cleave DNA poorly when their recognition sites are located close to DNA termini. Table 1.10 lists the activities of Fermentas restriction enzymes when their target sites are located close to the end of a PCR product.Experiments were performed as follows: PCR primers were designed with 1-5 extra
nucleotides at their 5’-end adjacent to the recognition site for the restriction enzyme. The 5’-end was labeled with [32P] by T4 Polynu-cleotide Kinase (#EK0031) and these labeled primers were used in the PCR reaction. PCR products were purified with the Silica Bead DNA Gel Extraction Kit (#K0513) and precipitated with
ethanol. The PCR products (0.5 µg) were incu-bated with 10 units of restriction enzymes in the optimal buffer for 1 hour at the recommended temperature. Reaction products were separated on 10% PAGE and the cleavage efficiency was determined using OptiQuant® Image Analysis software.
Cleavage Efficiency Close to the Termini of PCR Fragments
* – incubation was performed for 16 hours.Cleavage efficiency
0% 20-50% 0-20% 50-100%
172
1. C
ONVE
NTIO
NAL
REST
RICT
ION
ENZY
MES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Close vicinity of restriction targets within multiple cloning sites (MSC) of vectors may have negative effect on the efficiency of double digestions within MSC. For efficient double digestion within MSC the first reaction should be performed with a restriction enzyme that cleaves DNA inef-ficiently when enzyme’s target is located close to the end of DNA. The second digestion should
Cleavage of Restriction Targets Located in Close Vicinity within pUC19 Multiple Cloning Site
be performed with a restriction enzyme which tolerates a close proximity to the DNA end.Data presented below were generated using pUC19 plasmid DNA. The plasmid was cleaved with a primary restriction enzyme (the first cut) and end-labeled with [32P] by T4 Polynucle-otide Kinase (#EK0031). The DNA was then digested for one hour with a second restriction
enzyme (the second cut). Reaction products were separated by PAGE, and the amount of the label present on the DNA was determined by autoradiography. A decrease in radioactiv-ity reflects cleavage efficiency of the second restriction enzyme.
Table 1.11. Cleavage of restriction targets located in close vicinity within pUC19 multiple cloning site.
* – only double-stranded portion of DNA is included, not the overhangs.
– simultaneous double digestion may be performed with these enzyme pairs.
– this enzyme should be used for the first cut – this enzyme should be used for the second cut
– not recommended
For reaction conditions see www.fermentas.com/doubledigest
Enzyme pair First cutSecond cut
Efficiency of the 2nd
cut, %
bp from 1st cut*
Acc65I BamHI
Acc65I BamHI 100 4BamHI Acc65I 100 4
Acc65IEcl136II
Acc65I Ecl136II 100 1Ecl136II Acc65I 50-100 3
Acc65IEco24I
Acc65I Eco24I 100 1Eco24I Acc65I 0 1
Acc65IEcoRI
Acc65I EcoRI 100 7EcoRI Acc65I 100 7
Acc65ISacI
Acc65I SacI 50-100 1SacI Acc65I 0-20 1
Acc65IXapI
Acc65I XapI 50-100 7XapI Acc65I 50-100 7
BamHICfr9I
BamHI Cfr9I 50-100 0Cfr9I BamHI 50-100 0
BamHIEco88I
BamHI Eco88I 100 0Eco88I BamHI 50-100 0
BamHIHincII
BamHI HincII 50-100 7HincII BamHI 100 9
BamHIKpnI
BamHI KpnI 100 4KpnI BamHI 100 4
BamHISalI
BamHI SalI 50-100 7SalI BamHI 100 7
BamHIXbaI
BamHI XbaI 20-50 1XbaI BamHI 100 1
Cfr9IEcl136I
Cfr9I Ecl136II 50-100 5Ecl136II Cfr9I 100 7
Cfr9IEco24I
Cfr9I Eco24I 50-100 5Eco24I Cfr9I 100 5
Cfr9ISacI
Cfr9I SacI 50-100 5SacI Cfr9I 50-100 5
Cfr9IXbaI
Cfr9I XbaI 50-100 6XbaI Cfr9I 50-100 6
Ecl136IIEco88I
Ecl136II Eco88I 100 7Eco88I Ecl136II 50-100 5
Ecl136IIEcoRI
Ecl136II EcoRI 100 3EcoRI Ecl136II 100 1
Ecl136IIXapI
Ecl136II XapI 50-100 3XapI Ecl136II 50-100 1
Eco24IEco88I
Eco24I Eco88I 100 5Eco88I Eco24I 50-100 5
Eco24IEcoRI
Eco24I EcoRI 100 1EcoRI Eco24I 100 1
Eco24IKpnI
Eco24I KpnI 50-100 1KpnI Eco24I 0 1
Eco24IXapI
Eco24I XapI 50-100 1XapI Eco24I 20-50 1
Eco88ISacI
Eco88I SacI 100 5SacI Eco88I 50-100 5
Eco88IXbaI
Eco88I XbaI 100 6XbaI Eco88I 100 6
Enzyme pair First cutSecond cut
Efficiency of the 2nd
cut, %
bp from 1st cut*
EcoRICfr9I
EcoRI Cfr9I 50-100 11Cfr9I EcoRI 100 11
EcoRIEco88I
EcoRI Eco88I 50-100 11Eco88I EcoRI 100 11
EcoRIKpnI
EcoRI KpnI 100 7KpnI EcoRI 100 7
EcoRISacI
EcoRI SacI 50-100 1SacI EcoRI 20-50 1
HincIIPaeI
HincII PaeI 100 9PaeI HincII 100 7
HincIIPstI
HincII PstI 100 3PstI HincII 20-50 1
HincIISdaI
HincII SdaI 0-20 2SdaI HincII 20-50 1
HincIIXbaI
HincII XbaI 50-100 3XbaI HincII 50-100 1
HindIIIPaeI
HindIII PaeI 50-100 1PaeI HindIII 0 1
HindIIIPstI
HindIII PstI 100 7PstI HindIII 100 7
HindIIISdaI
HindIII SdaI 50-100 6SdaI HindIII 50-100 7
KpnIEcl136II
KpnI Ecl136II 50-100 1Ecl136II KpnI 100 3
KpnISacI
KpnI SacI 50-100 1SacI KpnI 100 1
KpnI XapI
KpnI XapI 100 7XapI KpnI 50-100 7
PaeIPstI
PaeI PstI 0 1PstI PaeI 20-50 1
PaeISalI
PaeI SalI 50-100 7SalI PaeI 100 7
PaeISdaI
PaeI SdaI 0 0SdaI PaeI 20-50 1
PstIBamHI
PstI BamHI 50-100 13BamHI PstI 50-100 13
PstISalI
PstI SalI 0 1SalI PstI 50-100 1
PstIXbaI
PstI XbaI 100 7XbaI PstI 50-100 7
SacISmaI
SacI SmaI 100 5SmaI SacI 50-100 7
SacIXapI
SacI XapI 0-20 1XapI SacI 50-100 1
SalISdaI
SalI SdaI 0 0SdaI SalI 20-50 1
SalIXbaI
SalI XbaI 50-100 1XbaI SalI 0-20 1
SdaIXbaI
SdaI XbaI 100 7XbaI SdaI 50-100 6
Enzyme pair First cutSecond cut
Efficiency of the 2nd
cut, %
bp from 1st cut*
SmaIAcc65I
SmaI Acc65I 0-20 1Acc65I SmaI 0 -1
SmaIBamHI
SmaI BamHI 50-100 2BamHI SmaI 0-20 0
SmaIEcl136II
SmaI Ecl136II 50-100 7Ecl136II SmaI 100 7
SmaIEco24I
SmaI Eco24I 50-100 7Eco24I SmaI 100 5
SmaIKpnI
SmaI KpnI 0 1KpnI SmaI 0 -1
SmaIXbaI
SmaI XbaI 50-100 8XbaI SmaI 100 6
pUC19 multiple cloning site
Eco24I Cfr9I HincII PstII XapI Ecl136II Acc65I Eco88I SalI BveI PaeI 396 EcoRI SacI KpnI SmaI BamHI XbaI XmiI SdaI HindIII 452 5’ - C CAG TGA ATT CGA GCT CGG TAC CCG GGG ATC CTC TAG AGT CGA CCT GCA GGC ATG CAA GCT TGG C - 3’ 3’ - G GTC ACT TAA GCT CGA GCC ATG GGC CCC TAG CAG ATC TCA GCT GGA CGT CCG TAC GTT CGA ACC G - 5’
173
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Site Preferences by Restriction Endonucleases
In 1975, Thomas and Davis discovered that EcoRI cleaves its five recognition sites on λ DNA at rates that differ by an order of magnitude (4). Similar examples have been documented for other restriction enzymes. Factors such as flanking sequences and the number of cleavage sites appear to influence cleavage efficiency (5). There are numerous restriction enzymes (EcoRII, NaeI, NarI, Ksp632I, BspMI, Eco57I, etc.), which never completely digest certain unmethylated target DNAs, even when using an excess of enzyme or prolonged incubation (6-9). Most of these enzymes are members of the expand-ing group of type II restriction enzymes which require simultaneous interaction with two copies of the target site for effective cleavage (10). These enzymes cleave DNA molecules with one recognition site very slowly. In the case of type IIE enzymes (EcoRII, NaeI), one of the target sequences serves as an allosteric effector for effective cleavage of the other recognition site (6, 11-13). Type IIF enzymes, (e.g., SfiI, Cfr10I, NgoMIV, BspMI) and majority of type IIB restriction enzymes (AlfI, AloI, BalI, BcgI, BsaXI, CspCI, FalI, PpiI, PsrI) cleave both recognition sequences in a concerted reaction (14-18). Type IIS enzymes, such as FokI, BpmI, BsgI and MboII, also interact with two copies of their recognition sequence before cleaving DNA by different mechanisms (19).Cleavage of resistant sites can be significantly enhanced by the addition of cleavable DNA, an oligodeoxyribonucleotide containing the recogni-tion site, or spermidine (7, 9, 17, 20, 21). Different restriction enzymes recognizing the same nucleotide sequence (isoschizomers or neoschizomers) often do not effectively cleave the same resistant recognition site (e. g., Fermentas enzymes BveI, Cfr42I, Eam1104I and PdiI and their prototypes BspMI, SacII, Ksp632I and NaeI). However, some isoschizomers or neoschizomers cleave “resistant” sites at the same rate as normal sites. For example, EheI cleaves target DNA more efficiently than its prototype NarI. Thus, one recognition site of NarI on λ DNA and two sites on pBR322 are not cleaved to completion, even after incubation with 50 units of the enzyme for 16 hours. Unlike NarI, Fermentas neoschizomer EheI cleaves λ DNA and pBR322 DNA completely under standard conditions.
Common PropertiesClassification of Restriction Enzymes
Restriction enzymes recognize specific nucleo-tide sequences and cleave DNA molecules either within or outside their recognition site. Digestion results in the generation of DNA with “sticky” (with 5’- or 3’-overhang) or “blunt” ends.
The phenomenon of host specificity was first observed by Luria and Human in the early 1950s (1). Nearly a decade later, Arber and Dussoix predicted its molecular basis (2). They proposed that host specificity is based on a two-enzyme system: a restriction enzyme, which recognizes specific DNA sequences and cleaves foreign DNA upon its entrance into the bacterial cell, and a modification enzyme (methyltransferase), which protects the host DNA from degradation by its own restriction enzyme. Both restriction and modification enzymes recognize the same nucleotide sequence and together they form a restriction-modification (R-M) system.
Restriction-modification systems are wide-spread among bacteria and have also been isolated from phage, Archaea and eukaryotic algae viruses. R-M systems have been classi-fied into four types (I, II, III and IV), depending on the complexity of their structure, cofactor requirements, mode of action and sequence cleaved (3). The best characterized and the most frequently used are type II restriction endonucleases. These enzymes recognize specific 4-8 bp DNA sequences and cleave a DNA sequence either within the recognition site, or at a specified position up to 20 base pairs outside the site.
Often there is more than one enzyme that recognizes a particular nucleotide sequence. According to restriction enzyme nomenclature, an enzyme with a unique specificity which has been discovered first is called the prototype. Subsequently discovered enzymes with the same specificity are termed isoschizomers. An isoschizomer may differ from the prototype enzyme in site preferences, reaction condi-tions, as well as in sensitivity to methylation and exhibition of star activity. Restriction enzymes that recognize the same nucleotide sequence, but cleave DNA at different posi-tions are called neoschizomers.
References1. Luria, S.E., Human, M.L., A nonhereditary, host-induced vari-
ation of bacterial viruses, J. Bacteriol., 64, 557-569, 1952.2. Arber, W., Dussoix, D., Host specifi city of DNA producted by
Escherichia Coli: I. Host controlled modifi cation of bacterio-phage lambda, J. Mol. Biol., 5, 18-36, 1962.
3. Roberts, R.J., et al., A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes, Nucleic Acids Res., 31, 1805-1812, 2003.
4. Thomas M., Davis R.W., Studies on the cleavage of bacte-riophage lambda DNA with EcoRI restriction endonucle-ase, J. Mol. Biol., 91, 315-328, 1975.
5. Sapienza P.J., et al., Thermodynamic and Kinetic Basis for the relaxed DNA Sequence Specificity of “Promiscuous” Mutant EcoRI endonuclease, J. Mol. Biol., 348, 307-324, 2005.
6. Kruger, D.H., et al., EcoRII can be activated to cleave refractory DNA recognition sites, Nucleic Acids Res., 16, 3997-4008, 1988.
7. Oller, A. R., et al., Ability of DNA and spermidine to affect the activity of restriction endonucleases from several bacterial species, Biochemistry, 30, 2543-2549, 1991.
8. Bolton, B.J., et al., Ksp632I: a novel class IIS restriction endonuclease from Kluyvera species 632 with the asym-metric hexanucleotide recognition sequence: 5’-CTCT-TCN↓-3’ 3’-GAGAAGNNNN↓-5’, Gene, 66, 31-43, 1988.
9. Reuter, M., et al., Use of specific oligonucleotide duplexes to stimulate cleavage of refractory DNA sites by restriction endonucleases, Anal. Biochem., 209, 232-237, 1993.
10. Halford, S.E., Hopping, jumping and looping by restriction endonucleases, Biochem. Soc. Trans, 29, 363-373, 2001.
11. Gabbara, S., Bhagwat, A.S., Interaction of EcoRII endonu-clease with DNA substrates containing single recognition sites, J.Biol.Chem., 267, 18623-18630, 1992.
12. Yang, C.C., Topal, M.D., Nonidentical DNA-binding sites of endonuclease NaeI recognize different families of sequences flanking the recognition site, Biochemistry, 31, 9657-9664, 1992.
13. Huai, Q., et al., Crystal structure of NaeI – an evolutionary bridge between DNA endonuclease and topoisomerase, EMBO J., 19, 3110-3118, 2000.
14. Wentzell, L.M., et al., The SfiI restriction endonuclease makes a four-strand DNA break at two copies of its recognition sequence, J. Mol. Biol., 248, 581-595, 1995.
15. Siksnys, V., et al., The Cfr10I restriction enzyme is func-tional as a tetramer, J. Mol. Biol., 291, 1105-1118, 1999.
16. Deibert, M., et al., Structure of the tetrameric restriction endonuclease NgoMIV in complex with cleaved DNA, Nat. Struct. Biol., 7, 792-799, 2000.
17. Gormley, N.A., et al., The type IIs restriction endonuclease BspMI is a tetramer that acts concertedly at two copies of an asymmetric DNA sequence, J. Biol. Chem., 277, 4034-4041, 2002.
18. Marshall, J.J., et al., Restriction Endonucleases that Bridge and lexcise two recognition sites from DNA, J. Mol. Biol., 367, 419-431, 2007.
19. Bath, A.J., et al., Many type IIs restriction endonucleases interact with two recognition sites before cleaving DNA, J. Biol. Chem., 277, 4024-4033, 2002.
20. Conrad, M., Topal, M.D., DNA and spermidine provide a switch mechanism to regulate the activity of restriction enzyme NaeI, Proc. Natl.Acad Sci. USA, 86, 9707-9711, 1989.
21. Grigaite, R.J., et al., AarI, a restriction endonuclease from Arthrobacter aurescens SS2-322, which recognizes the novel non-palindromic sequence 5’-CACCTGC(N)4/8-3’, Nucleic Acids Res., 30, e123, 2002.
174
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
References1. Nasri, M., Thomas, D., Relaxation of recognition sequence
of specific endonuclease HindIII, Nucleic Acids Res., 14, 811-821, 1986.
2. George, J., Chirikjian, J.G., Sequence-specific endonu-clease BamHI: Relaxation of sequence recognition, Proc. Natl. Acad. Sci. USA, 79, 2432-2436, 1982.
3. Kolesnikov, V.A., et al., Relaxed specificity of endonuclease BamHI as determined by identification of recognition sites in SV40 and pBR322 DNAs, FEBS Letters, 132, 101-103, 1981.
4. Polisky, B., et al., Specificity of substrate recognition by the EcoRI restriction endonuclease, Proc. Natl. Acad. Sci. USA, 72, 3310-3314, 1975.
5. Woodbury, C.P., et al., DNA site recognition and reduced specificity of the EcoRI endonuclease, J. Biol. Chem., 255, 11534-11546, 1980.
6. George, J., et al., Sequence-specific endonuclease BamHI, J. Biol. Chem., 255, 6521-6524, 1980.
7. Malyguine, E., et al., Alteration of the specificity of restric-tion endonucleases in the presence of organic solvents, Gene, 8, 163-177, 1980.
8. Hsu, M., Berg, P., Altering the specificity of restriction endonuclease: effect of replacing Mg2+ with Mn2+, Biochemistry, 17, 131-138, 1978.
9. Mayer, H., Optimization of the EcoRI* activity of EcoRI endonuclease, FEBS Letters, 90, 341-344, 1978.
10. Nasri, M., Thomas, D., Alteration of the specificity of PvuII restriction endonuclease, Nucleic Acids Res., 15, 7677-7687, 1987.
11. Bitinaite, J., Schildkraut, I., Self generated DNA termini relax the specificity of SgrAI restriction endonuclease, Proc. Natl. Acad. Sci. USA, 99, 1164-1169, 2002.
Star Activity
Restriction enzymes recognize specific nucleo-tide sequences within DNA molecules. However, their recognition site specificity can be reduced in vitro (1). Under certain conditions, enzymes are able to recognize and cleave nucleotide sequences which differ from the canonical site. At low ionic strength, for example, BamHI (with the recognition sequence GGATCC) is able to cleave the following sequences: NGATCC, GPuATCC and GGNTCC (2, 3). This phenomenon is called “relaxed” or “star” activity (4, 5). For most restriction enzyme applications, star activity is not desirable.
Reports (4, 6-10) on star activity have sug-gested that this phenomenon is the result of:
• prolonged incubation (over digestion), • high enzyme concentration in the reaction
mixture (over digestion), • high glycerol concentration in the reaction
mixture,• presence of organic solvents, such as etha-
nol, dimethyl sulfoxide (DMSO) or dimethyl formamide (DMFA), in the reaction mixture,
• low ionic strength of the reaction buffer,• suboptimal pH values of the reaction buffer,• substitution of Mg2+ for other divalent
cations, such as Mn2+ or Co2+.
In some cases, the termini generated by DNA cleavage with a restriction enzyme at the canonical site have been shown to stimulate enzyme star activity (11).Both star activity and incomplete DNA digestion result in atypical electrophoresis patterns that can be distinguished by careful examination of gel images (see Fig. 1.4). Any tendency of a restriction enzyme to exhibit star activity is indicated both in the product description and in the Certificate of Analysis.
Figure 1.4. Enzyme complete digestion and star activity.1 – Lambda DNA.2 – Lambda DNA incubated 1 hour with 0.15 u of EcoRI (incomplete cleavage).3 – Lambda DNA incubated 1 hour with 0.4 u of EcoRI (incomplete cleavage).4 – Lambda DNA incubated 1 hour with 1 u of EcoRI (complete digestion).5 – Lambda DNA incubated 16 hours with 40 u of EcoRI (star activity).6 – Lambda DNA incubated 16 hours with 70 u of EcoRI (star activity).
How to distinguish between star activity and incomplete digestion?
• Star activity results in additional DNA bands below the expected bands and no additional bands above the largest expected fragment. These additional bands become more intense, while the expected bands become less intense, when either the incubation time or the amount of enzyme is increased.
• Incomplete DNA digestion results in additional bands above the expected DNA bands on the gel. Additional bands disap-pear when the incubation time or amount of enzyme is increased. No additional bands below the smallest expected frag-ment are observed.
NoteCertain restriction enzymes remain associated with the substrate DNA after cleavage and cause a change in the mobility of the digestion products during electrophoresis. The resulting atypical pattern is not related to star activity. In these cases, use a loading dye with SDS (6X DNA Loading Dye & SDS Solution, #R1151), heat the sample for 10 min at 65°C and chill on ice prior to loading on the gel (see p.433). This effect is characteristic of the following
FastDigest® enzymes:FastDigest® BspMI (BveI), FastDigest® HgaI (CseI), FastDigest® AcuI (Eco57I), FastDigest® FokI, FastDigest® MboII, FastDigest® TauI, FastDigest® TspRI (TscAI) and
Conventional restriction enzymes:AarI, AloI, BdaI, BseXI (BbvI), BveI (BspMI), CseI (HgaI), Eco57I (AcuI), Eco57MI, EcoRII, FaqI (BsmFI), GsuI (BpmI), Lsp1109I (BbvI), LweI (SfaNI), MboII, MnlI, SchI (MlyI), TauI, TscAI (TspRI), TsoI, TstI.
Incomplete cleavage Star activity Complete digestion
1 2 3 4 5 6
175
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Digestion of Methylated DNA
DNA methylation is the process of transferring a methyl group from a donor molecule to either a cytosine or an adenine by DNA methyltrans-ferases. Such methylation is the most common and abundant DNA modification process in living organisms. Three types of methylated bases are predominantly found in DNA:
5-methylcytosine (m5C), N4-methylcytosine (m4C), N6-methyladenine (m6A).
Other modified bases, such as 5-hydroxy-methylcytosine (hm5C) and 5-hydroxymethy-luracil (hm5U), have also been described. The organism-specific pattern of methylation depends on the methyltransferases’ specificity.In prokaryotes, DNA cleavage by a cognate restriction enzyme is prevented by the methyla-tion of DNA by a sequence-specific methyltrans-ferase which is an integral component of every restriction-modification (R-M) system (4, 5).The majority of E.coli strains used for propagation of plasmid DNA contain two site-specific DNA methyltransferases – Dam and Dcm (6, 4). The methylase encoded by the dam gene methylates the N6-position of an adenine residue within the GATC sequence (8, 9). The methylase encoded by the dcm gene methylates the C5-position of the internal cytosine residue within the CCWGG sequence (7, 10). In addition to Dam and Dcm methylases, labora-tory strains of E.coli K12 and B may contain EcoKI or EcoBI enzymes, respectively, encoded by a type I R-M system. These methyltransferases modify adenine residues within their respective recognition sequences: AAC(N)6GTGC for EcoKI and TGA(N)8TGCT for EcoBI (3, 4).
References1. Luria, S.E., Human, M.L., A nonhereditary, host-induced varia-
tion of bacterial viruses, J. Bacteriol., 64, 557-569, 1952.2. Arber, W., Dussoix, D., Host specifi city of DNA producted
by Escherichia Coli: I. Host controlled modifi cation of bacteriophage lambda, J. Mol. Biol., 5, 18-36, 1962.
3. Roberts, R.J., et al., A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes, Nucleic Acids Res., 31, 1805-1812, 2003.
4. McClelland, M., The effect of sequence specific DNA me-thylation on restriction endonuclease cleavage, Nucleic Acids Res., 9, 5859-5866, 1981.
5. McClelland, M., et al., Effect of site-specific modification on restriction endonucleases and DNA modification meth-yltransferases, Nucleic Acids Res., 22, 3640-3659, 1994.
6. Marinus, M.G., Morris, N.R., Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12, J. Bacte-riol., 114, 1143-1150, 1973.
7. May, M.S., Hattman, S., Analysis of bacteriophage deoxy-ribonucleic acid sequences methylated by host- and R-factor-controlled enzymes, J. Bacteriol., 123, 768-770, 1975.
8. Hattman, S., et al., Sequence specificity of the P1 modi-fication methylase (M.EcoP1) and the DNA methylase (M.Ecodam) controlled by the Escherichia coli dam gene, J. Mol. Biol.,126, 367-380, 1978.
9. Geier, G.E., Modrich, P., Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of DpnI endonuclease, J. Biol. Chem., 254, 1408-1413, 1979.
10. Buryanov, Ya.I., et al., Site specificity and chromatographic properties of E.coli K12 and EcoRII DNA-cytosine methy-lases, FEBS Letters, 88, 251-254, 1978.
11. Waalwijk, C., Flavell, R.A., MspI, an isochizomer of HpaII which cleaves both unmethylated and methylated HpaII sites, Nucleic Acids Res., 5, 3231-3236, 1978.
12. Bird, A.P., et al., Methylated and unmethylated DNA com-partments in the sea urchin genome, Cell, 17, 889-902, 1979.
13. McClelland, M., The frequency and distribution of methylat-able DNA sequences in leguminous plant protein coding genes, J. Mol. Evol., 19, 346-354, 1983.
14. Dreiseikelmann, B., et al., The effect of differential methy-lation by Escherichia coli of plasmid DNA and phage T7 and lambda DNA on the cleavage by restriction endonuclease MboI from Moraxella bovis, Biochim. Biophys. Acta, 562, 418-428, 1979.
15. Butkus, V. et al., Investigation of restriction-modification enzymes from M.varians RFL19 with a new type of speci-ficity toward modification of substrate, Nucleic Acids Res., 13, 5727-5746, 1985.
DNA from higher eukaryotic organisms pos-sesses modified 5-methylcytosine residues within CpG or CpNpG contexts (5, 11-13). These tissue-specific methylation patterns are heritable. They participate in regulation of gene expression and cellular differentiation. In cases where a restriction enzyme target site overlaps a methylation site, the following diges-tion results are possible:
• no effect• partial inhibition• complete block
The ability to cleave methylated DNA is an intrinsic and unpredictable property of each restriction enzyme. Therefore, isoschizomers and neoschizomers which recognize the same DNA sequences can differ in their sensitivity to DNA methylation (Table 1.12). For instance, MboI (recognition sequence GATC) does not cleave DNA methylated by Dam methylase (14), while the isoschizomer Bsp143I is insensitive to Dam methylation. EcoRII does not cleave DNA at the CCWGG site if it is methylated by Dcm, while its neoisoschizomer MvaI will cleave this methylated site (15). Thus, to achieve effective DNA diges-tion, it is necessary to take into account both the type of DNA methylation and the sensitivity of the restriction enzyme to that type of methylation.All restriction enzymes produced by Fermentas have been examined for their sensitivity to Dam, Dcm, CpG, EcoKI and EcoBI methylation of substrate DNA. Detailed methylation sensitivity information for our restriction enzymes is listed in the tables on pp.176-181, as well as in the pro-duct descriptions and Certificates of Analysis.
Conventional restriction enzyme couple
Recognition and cleavage sites
Sensitivity to methylation
Acc65I (Asp718I) G↓GTACC Overlapping Dcm or CpG methylation may influence DNA cleavageKpnI GGTAC↓C Not influenced by Dcm or CpG methylationApaI GGGCC↓C Overlapping Dcm or CpG methylation may influence DNA cleavageBsp120I (PspOMI) G↓GGCCC Blocked by overlapping Dcm or CpG methylationBsp143I (Sau3AI) ↓GATC Not influenced by Dam, blocked by CpG methylationMboI ↓GATC Blocked by Dam methylated DNADpnI GA↓TC Cleaves only Dam methylated DNABspOI (BmtI) GCTAG↓C Not influenced by CpG methylationNheI G↓CTAGC Overlapping CpG methylation may influence DNA cleavageCfr9I (XmaI) C↓CCGGG CpG methylation may influence DNA cleavageSmaI CCC↓GGG Blocked by CpG methylationCsp6I (CviQI) G↓TAC Not influenced by CpG methylationRsaI GT↓AC Overlapping CpG methylation may influence DNA cleavageEcl136II (EcoICRI) GAG↓CTC Overlapping CpG methylation may influence DNA cleavageSacI GAGCT↓C Not influenced by CpG methylationEcoRII ↓CCWGG Blocked by Dcm methylationMvaI (BstNI) CC↓WGG Not influenced by Dcm methylationHpaII C↓CGG Blocked by CpG methylationMspI (HpaII) C↓CGG Not influenced by CpG methylation
Table 1.12. Fermentas isoschizomers and neoschizomers with differing sensitivities to target methylation.
Single letter codeR = G or A; H = A, C or T; Y = C or T; V = A, C or G;W = A or T; B = C, G or T;M = A or C; D = A, G or T;K = G or T; N = G, A, T or C.S = C or G;
176
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Note** – DpnI (Gm6A↓TC) – cleaves only Dam methylated DNA.
* – recognition sequence is indicated in bold.
m6A = N6-methyladenine.
– overlapping methylase sequences.
– cleavage not blocked.
– cleavage blocked.
– cleavage rate is slowed significantly by methylation.
?– the sensitivity to methylation has not been determined.
Single letter codeR = G or A; H = A, C or T; Y = C or T; V = A, C or G;W = A or T; B = C, G or T;M = A or C; D = A, G or T;K = G or T; N = G, A, T or C.S = C or G;
Table 1.14. Partially overlapping Dam methyltrans-ferase and restriction enzyme recognition sites.
?
?
?
Conventional FastDigest® Sequence* Effect
AloI5’...GAAC(N)4Gm6A TCC...3’
3’...CTTG(N)4C Tm6AGG...5’
BdaI5’...TGm6A TC(N)4TCA...3’
3’...AC Tm6AG(N)4AGT...5’
BseJIBsaBI
5’...Gm6A TC(N)3ATC...3’
3’...C Tm6AG(N)3TAG...5’
Bsh1285IBsiEI
5’...CGm6A TCG...3’
3’...GC Tm6AGC...5’
Bsp68INruI
5’...Gm6A TCGCGm6A TC...3’
3’...C Tm6AGCGC Tm6AG...5’
Bsu15I ClaI
5’...ATCGm6A TC...3’
3’...TAGC Tm6AG...5’
Hin4I
5’...Gm6A TC(N)4VTC...3’
3’...C Tm6AG(N)4BAG...5’
5’...GAY(N)4Gm6A TC...3’
3’...CTR(N)4C Tm6AG...5’
HphI5’...GGTGm6A TC...3’
3’...CCAC Tm6AG...5’
Kpn2I Kpn2I
5’...TCCGGm6A TC...3’
3’...AGGCC Tm6AG...5’
5’...Gm6A TCCGGm6A TC...3’
3’...C Tm6AGGCC Tm6AG...5’
MboII MboII
5’...GAAGm6A TC...3’
3’...CTTC Tm6AG...5’
PagIBspHI
5’...TCATGm6A TC...3’
3’...AGTAC Tm6AG...5’
5’...Gm6A TCATGm6A TC...3’
3’...C Tm6AGTAC Tm6AG...5’
PfoI PfoI
5’...TCCNGGm6A TC...3’
3’...AGGNCC Tm6AG...5’
SmoI5’...CTYRAGm6A TC...3’
3’...GARYTC Tm6AG...5’
TaqI TaqI
5’...TCGm6A TC...3’
3’...AGC Tm6AG...5’
TstI5’...CAC(N)4Gm6A TCC...3’
3’...GTC(N)4C Tm6AGG...5’
XbaI XbaI
5’...TCTAGm6A TC...5’
3’...AGATC Tm6AG...5’
Effect of Dam Methylation on DNA Cleavage
To cleave with a restriction enzyme which is sensitive to the Dam methylation, DNA should be purified from dam– E.coli strains. E.coli GM2163 dam–, dcm– (#M0099) is available upon request with any product purchase. Control digestions should be performed with Lambda DNA (dam–, dcm–), #SD0021.
Table 1.13. Completely overlapping Dam methyl-transferase and restriction enzyme recognition sites.
Conventional FastDigest® Sequence* Effect
BamHI BamHI
GGm6ATCC
BclIBclI
TGm6ATCA
BglIIBglII
AGm6ATCT
Bsp143ISau3AI
Gm6ATC
BspPI GGm6ATC
DpnI**DpnI
Gm6ATC
MboIMboI
Gm6ATC
PsuIPsuI
RGm6ATCY
PvuIPvuI
CGm6ATCG
SfaAIAsiSI
GCGm6ATCGC
177
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Effect of Dcm Methylation on DNA Cleavage
To cleave with a restriction enzyme which is sensitive to Dcm methylation, DNA should be purified from dcm– E.coli strains. E.coli GM2163 dam–, dcm– (#M0099) is available upon request with any product purchase. Control digestions should be performed with Lambda DNA (dam–, dcm–), #SD0021.
Table 1.15. Completely overlapping Dcm methyl-transferase and restriction enzyme recognition sites.
Conventional FastDigest® Sequence* Effect
EcoRII Cm5CWGG
MvaI MvaI
Cm5CWGG
PasI CCm5CWGGG
SexAI ACm5CWGGT
SgeI** Cm5CWGG
Table 1.16. Partially overlapping Dcm methyltrans-ferase and restriction enzyme recognition sites.
Note* – recognition sequence is indicated in bold.
** – SgeI cleaves only methylated DNA.
– overlapping methylase sequences. m5C = 5-methylcytosine.
– cleavage not blocked.
– cleavage blocked.
– cleavage rate is slowed significantly by methylation.
Single letter codeR = G or A; H = A, C or T; Y = C or T; V = A, C or G;W = A or T; B = C, G or T;M = A or C; D = A, G or T;K = G or T; N = G, A, T or C.S = C or G;
Conventional FastDigest® Sequence* Effect
Acc65IAcc65I
5’...GGTACm5CW GG...3’
3’...CCATG GWm5CC...5’
5’...Cm5CW GGTACm5CW GG...3’
3’...G GWm5CCATG GWm5CC...5’
AloI5’...GAAC(N)6TCm5CW GG...3’
3’...CTTG(N)6AG GWm5CC...5’
ApaI ApaI
5’...GGGCCm5CW GG...3’
3’...CCCGG GWm5CC...5’
BamHIBamHI
5’...Cm5CW GGATCm5CW GG...3’
3’...G GWm5CCTAG GWm5CC...5’
BfuI5’...GTATCm5CW GG...3’
3’...CATAG GWm5CC...5’
BglI BglI
5’...GCm5CW GGNNGGC...3’
3’...CG GWm5CCNNCCG...5’
Bme1390I ScrFI
5’...Cm5CW GG...3’
3’...G GWm5CC...5’
BseDI BsaJI
5’...Cm5CW GGG...3’
3’...G GWm5CCC...5’
BseGI BseGI
5’...Cm5CW GGATG...3’
3’...G GWm5CCTAC...5’
BseLI BslI
5’...Cm5CW GG(N)4GG...3’
3’...G GWm5CC(N)4CC...5’
5’...Cm5CW GGNCm5CW GG...3’
3’...G GWm5CCNG GWm5CC...5’
BseSI Bme1580I
5’...GKGCCm5CW GG...3’
3’...CMCGG GWm5CC...5’
BshNI BanI
5’...GGYRCm5CW GG...3’
3’...CCRYG GWm5CC...5’
5’...Cm5CW GGYRCm5CW GG...3’
3’...G GWm5CCRYG GWm5CC...5’
Bsp120I Bsp120I
5’...GGGCCm5CW GG...3’
3’...CCCGG GWm5CC...5’
BspLI NlaIV
5’...GGNNCm5CW GG...3’
3’...CCNNG GWm5CC...5’
5’...Cm5CW GGNNCm5CW GG...3’
3’...G GWm5CCNNG GWm5CC...5’
BspPI5’...Cm5CW GGATC...3’
3’...G GWm5CCTAG...5’
BstXI BstXI
5’...Cm5CA GG(N)4TGG...3’
3’...G GTm5CC(N)4ACC...5’
5’...Cm5CA GGNNCm5CT GG...3’
3’...G GTm5CCNNG GAm5CC...5’
BsuRI HaeIII
5’...GGCm5CW GG...3’
3’...CCG GWm5CC...5’
5’...Cm5CW GGCm5CW GG...3’
3’...G GWm5CCG GWm5CC...5’
CaiI AlwNI
5’...CAGNNCm5CT GG...3’
3’...GTCNNG GTm5CC...5’
CfrI5’...YGGCm5CW GG...3’
3’...RCCG GWm5CC...5’
Cfr13I Sau96I
5’...GGNCm5CW GG...3’
3’...CCNG GWm5CC...5’
Eco24I5’...Cm5CW GGGCCm5CW GG...3’
3’...G GWm5CCCGG GWm5CC...5’
Conventional FastDigest® Sequence* Effect
Eco31I Eco31I
5’...Cm5CW GGTCTC...3’
3’...G GWm5CCAGAG...5’
Eco47I AvaII
5’...GGWCm5CW GG...3’
3’...CCWG GWm5CC...5’
Eco91I Eco91I
5’...Cm5CW GGTNACm5CW GG...3’
3’...G GWm5CCANTG GWm5CC...5’
Eco57MI5’...Cm5CT GGAG...3’
3’...G GAm5CCTC...5’
Eco147I StuI
5’...AGGCm5CT GG...3’
3’...TCCG GAm5CC...5’
EcoO109IEcoO109I
5’...RGGNCm5CT GG...3’
3’...YCCNG GAm5CC...5’
EheI EheI
5’...Cm5CW GGCGCm5CW GG...3’
3’...G GWm5CCGCG GWm5CC...5’
FaqI BsmFI
5’...Cm5CW GGGAC...3’
3’...G GWm5CCCTG...5’
FokI5’...Cm5CW GGATG...3’
3’...G GWm5CCTAC...5’
GsuI BpmI
5’...CTCm5CA GG...3’
3’...GAG GTm5CC...5’
Hin1I BsaHI
5’...GRCGCm5CW GG...3’
3’...CYGCG GWm5CC...5’
HphI5’...Cm5CW GGTGA...3’
3’...G GWm5CCACT...5’
KpnI KpnI
5’...GGTACm5CW GG...3’
3’...CCATG GWm5CC...5’
5’...Cm5CW GGTACm5CW GG...3’
3’...G GWm5CCATG GWm5CC...5’
MlsI MscI
5’...TGGCm5CA GG...3’
3’...ACCG GTm5CC...5’
PfoI PfoI
5’...TCm5CA GGA...3’
3’...AG GTm5CCT...5’
Psp5II PpuMI
5’...RGGWCm5CT GG...3’
3’...YCCWG GAm5CC...5’
SduI Bsp1286I
5’...Cm5CW GGGCCm5CW GG...3’
3’...G GWm5CCCGG GWm5CC...5’
SfiI SfiI
5’...GGCm5CW GGNNGGCC...3’
3’...CCG GWm5CCNNCCGG...5’
5’...Cm5CW GGCC(N)5GGCm5CW GG...3’
3’...G GWm5CCGG(N)5CCG GWm5CC...5’
SgsI AscI
5’...Cm5CW GGCGCGCm5CW GG...3’
3’...G GWm5CCGCGCG GWm5CC...5’
SspDI5’...Cm5CW GGCGCC...3’
3’...G GWm5CCGCGG...5’
TsoI5’...TARCm5CA GG...3’
3’...ATYG GTm5CC...5’
TstI5’...CAC(N)6TCm5CW GG...3’
3’...GTG(N)6AG GWm5CC...5’
Van91I PflMI
5’...Cm5CA GG(N)3TGG...3’
3’...G GTm5CC(N)3ACC...5’
XagI EcoNI
5’...Cm5CT GG(N)3AGG...3’
3’...G GAm5CC(N)3TCC...5’
5’...Cm5CT GGNCm5CA GG...3’
3’...G GAm5CCNG GTm5CC...5’
178
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.17. CpG is located inside the recognition site.
Effect of CpG Methylation on DNA Cleavage
Methylated DNA substrates were prepared using SssI methyltransferase.
ConventionalFastDigest® Sequence Effect
HpaII HpaII
CCGG
Kpn2I Kpn2I
TCCGGA
MauBIMauBI
CGCGCGCG
MbiI BsrBI
GAGCGG
MluI MluI
ACGCGT
MreI MreI
CGCCGGCG
MspI MspI
CCGG
NotI NotI
GCGGCCGC
NsbI FspI
TGCGCA
PauIBssHII
GCGCGC
PdiINaeI
GCCGGC
Pfl23IIBsiWI
CGTACG
Ppu21IBsaAI
YACGTR
Psp1406I AclI
AACGTT
PvuI PvuI
CGATCG
SalI SalI
GTCGAC
SfaAI AsiSI
GCGATCGC
SgrDI CGTCGACG
SgsI AscI
GGCGCGCC
SmaI SmaI
CCCGGG
SmuI CCCGC
SsiI AciI
CCGC
SspDI GGCGCC
TaiI TaiI
ACGT
TaqI TaqI
TCGA
TauI GCSGC
XhoI XhoI
CTCGAG
ConventionalFastDigest® Sequence Effect
AatIIAatII
GACGTC
AjiI CACGTC
BauI CACGAG
BcnINciI
CCSGG
Bsh1236IBsh1236I
CGCG
Bsh1285I BsiEI
CGRYCG
BshTI AgeI
ACCGGT
Bsp68INruI
TCGCGA
Bsp119I Bsp119I
TTCGAA
Bsu15I ClaI
ATCGAT
Cfr9I CCCGGG
Cfr10I BsrFI
RCCGGY
Cfr42I CCGCGG
CpoIRsrII
CGGWCCG
CseIHgaI
GACGC
Eco47III AfeI
AGCGCT
Eco52I EagI
CGGCCG
Eco72I PmlI
CACGTG
Eco88I AvaI
CYCGRG
Eco105I SnaBI
TACGTA
EheI EheI
GGCGCC
Esp3IBsmBI
CGTCTC
FspAI FspAI
RTGCGCAY
HaeII RGCGCY
HhaI HhaI
GCGC
Hin1I BsaHI
GRCGYC
Hin6I HinP1I
GCGC
Note
– cleavage not blocked.
– cleavage blocked.
– cleavage rate is reduced significantly by methylation.
Single letter codeR = G or A; H = A, C or T; Y = C or T; V = A, C or G;W = A or T; B = C, G or T;M = A or C; D = A, G or T;K = G or T; N = G, A, T or C.S = C or G;
179
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.18. CpG partially overlaps the recognition site.
Conventional FastDigest® Sequence* Effect
AarI5’...CACCTGm5C G...3’
3’...GTGGAC Gm5C...5’
AasIDrdI
5’...m5C GAC(N)6GTC...3’
3’... Gm5CTG(N)6CAG...5’
5’...m5C GAC(N)6GTm5C G...3’
3’... Gm5CTG(N)6CA Gm5C...5’
5’...GAm5C G(N)5GTC...3’
3’...CT Gm5C(N)5CAG...5’
5’...GAm5C G(N)4m5C GTC...3’
3’...CT Gm5C(N)4 Gm5CAG...5’
Acc65I Acc65I
5’...GGTACm5C G...3’
3’...CCATG Gm5C...5’
5’...m5C GGTACm5C G...3’
3’... Gm5CCATG Gm5C...5’
AdeI DraIII
5’...CAm5C GNNGTG...3’
3’...GT Gm5CNNCAC...5’
5’...CAm5C GNm5C GTG...3’
3’...GT Gm5CN Gm5CAC...5’
AjuIAjuI
5’...m5C GAA(N)7TTGG...3’
3’... Gm5CTT(N)7AACC...5’
AlfI5’...GCA(N)6TGm5C G...3’
3’...CGT(N)6AC Gm5C...5’
AloI
5’...m5C GAAC(N)6TCC...3’
3’... Gm5CTTG(N)6AGG...5’
5’...GAAC(N)6TCm5C G...3’
3’...CTTG(N)6AG Gm5C...5’
5’...GAAm5C G(N)5TCC...3’
3’...CTT Gm5C(N)5AGG...5’
Alw21I Alw21I
5’...m5C GWGCWm5C G...3’
3’... Gm5CWCGW Gm5C...5’
Alw26I Alw26I
5’...GTCTm5C G...3’
3’...CAGA Gm5C...5’
5’...m5C GTCTC...3’
3’... Gm5CAGAG...5’
Alw44I ApaLI
5’...GTGCAm5C G...3’
3’...CACGT Gm5C...5’
ApaI ApaI
5’...GGGCCm5C G...3’
3’...CCCGG Gm5C...5’
5’...m5C GGGCCm5C G...3’
3’... Gm5CCCGG Gm5C...5’
BamHI BamHI
5’...m5C GGATCm5C G...3’
3’... Gm5CCTAG Gm5C...5’
BfuI
5’...m5C GTATCC...3’
3’... Gm5CATAGG...5’
5’...GTATCm5C G...3’
3’...CATAG Gm5C...5’
BglI BglI
5’...GCm5C G(N)3m5C GGC...3’
3’...CG Gm5C(N)3 Gm5CCG...5’
5’...GCC(N)5GGm5C G...3’
3’...CGG(N)5CC Gm5C...5’
5’...m5C GCC(N)5GGm5C G...3’
3’... Gm5CGG(N)5CC Gm5C...5’
Bme1390I ScrFI
5’...Cm5C GGG...3’
3’...G Gm5CCC...5’
?
?
Conventional FastDigest® Sequence* Effect
BoxI PshAI
5’...GAm5C G(N)3GTC...3’
3’...CT Gm5C(N)3CAG...5’
5’...GAm5C GNNm5C GTC...3’
3’...CT Gm5CNN Gm5CAG...5’
5’...GAC(N)4GTm5C G...3’
3’...CTG(N)4CA Gm5C...5’
BpiI BbsI
5’...GAAGAm5C G...3’
3’...CTTCT Gm5C...5’
5’...m5C GAAGAC...3’
3’... Gm5CTTCTG...5’
BplI BplI
5’...m5C GAG(N)5CTm5C G...3’
3’... Gm5CTC(N)5GA Gm5C...5’
Bpu10I Bpu10I
5’...CCTNAGm5C G...3’
3’...GGANTC Gm5C...5’
Bpu1102I BlpI
5’...m5C GCTNAGm5C G...3’
3’... Gm5CGANTC Gm5C...5’
BseDI BsaJI
5’...Cm5C Gm5C GG...3’
3’...G Gm5C Gm5CC...5’
BseGI BseGI
5’...m5C GGATG...3’
3’... Gm5CCTAC...5’
BseJIBsaBI
5’...GAT(N)4ATm5C G...3’
3’...CTA(N)4TA Gm5C...5’
5’...m5C GAT(N)4ATm5C G...3’
3’... Gm5CTA(N)4TA Gm5C...5’
BseLI BslI
5’...Cm5C G(N)6GG...3’
3’...G Gm5C(N)6CC...5’
5’...Cm5C G(N)5m5C GG...3’
3’...G Gm5C(N)5 Gm5CC...5’
BseMIBsrDI
5’...m5C GCAATG...3’
3’... Gm5CGTTAC...5’
BseSIBme1580I
5’...m5C GKGCMm5C G...3’
3’... Gm5CMCGK Gm5C...5’
BseXIBseXI
5’...m5C GCAGm5C G...3’
3’... Gm5CGTC Gm5C...5’
BshNI BanI
5’...GGYRCm5C G...3’
3’...CCRYG Gm5C...5’
5’...m5C GGYRCm5C G...3’
3’... Gm5CCRYG Gm5C...5’
5’...GGm5C GCC...3’
3’...CC Gm5CGG...5’
Bsp120I Bsp120I
5’...GGGCCm5C G...3’
3’...CCCGG Gm5C...5’
Bsp143I Sau3AI
5’...GATm5C G...3’
3’...CTA Gm5C...5’
BspLI NlaIV
5’...GGNNCm5C G...3’
3’...CCNNG Gm5C...5’
5’...m5C GGNNCm5C G...3’
3’... Gm5CCNNG Gm5C...5’
BspOI BmtI
5’...GCTAGm5C G...3’
3’...CGATC Gm5C...5’
5’...m5C GCTAGm5C G...3’
3’... Gm5CGATC Gm5C...5’
BspPI
5’...GGATm5C G...3’
3’...CCTA Gm5C...5’
5’...m5C GGATC...3’
3’... Gm5CCTAG...5’
?
(continued on next page)
Conventional FastDigest® Sequence* Effect
Bst1107I BstZ17I
5’...GTATAm5C G...3’
3’...CATAT Gm5C...5’
5’...m5C GTATAm5C G...3’
3’... Gm5CATAT Gm5C...5’
BsuRI HaeIII
5’...m5C GGCm5C G...3’
3’... Gm5CCG Gm5C...5’
BveIBspMI
5’...ACCTGm5C G...3’
3’...TGGAC Gm5C...5’
CfrI5’...YGGCm5C G...3’
3’...RCCG Gm5C...5’
Cfr13I Sau96I
5’...GGNCm5C G...3’
3’...CCNG Gm5C...5’
Csp6I Csp6I
5’...m5C GTAm5C G...3’
3’... Gm5CAT Gm5C...5’
DpnI DpnI
5’...Gm6A Tm5C G...3’
3’...C Tm6A Gm5C...5’
Eam1104IEarI
5’...CTCTTm5C G...3’
3’...GAGAA Gm5C...5’
Eam1105I Eam1105I
5’...GAm5C G(N)3m5C GTC...3’
3’...CT Gm5C(N)3 Gm5CAG...5’
5’...GAC(N)5GTm5C G...3’
3’...CTG(N)5CA Gm5C...5’
5’...m5C GAC(N)5GTm5C G...3’
3’... Gm5CTG(N)5CA Gm5C...5’
Ecl136II Ecl136II
5’...GAGCTm5C G...3’
3’...CTCGA Gm5C...5’
5’...m5C GAGCTm5C G...3’
3’... Gm5CTCGA Gm5C...5’
Eco24I5’...m5C GRGCYm5C G...3’
3’... Gm5CYCGR Gm5C...5’
Eco31I Eco31I
5’...GGTCTm5C G...3’
3’...CCAGA Gm5C...5’
5’...m5C GGTCTC...3’
3’... Gm5CCAGAG...5’
Eco32I EcoRV
5’...m5C GATATm5C G...3’
3’... Gm5CTATA Gm5C...5’
Eco47I AvaII
5’...GGWCm5C G...3’
3’...CCWG Gm5C...5’
Eco91I Eco91I
5’...m5C GGTNACm5C G...3’
3’... Gm5CCANTG Gm5C...5’
EcoRI EcoRI
5’...m5C GAATTC...3’
3’... Gm5CTTAAG...5’
5’...m5C GAATTm5C G...3’
3’... Gm5CTTAA Gm5C...5’
FaqIBsmFI
5’...GGGAm5C G...3’
3’...CCCT Gm5C...5’
5’...m5C GGGAC...3’
3’... Gm5CCCTG...5’
FokI5’...m5C GGATG...3’
3’... Gm5CCTAC...5’’
Hin4I
5’...m5C GAY(N)5VTC...3’
3’... Gm5CTR(N)5BAG...5’
5’...GAY(N)5VTm5C G...3’
3’...CTR(N)5BA Gm5C...5’
5’...m5C GAY(N)5VTm5C G...3’
3’... Gm5CTR(N)5BA Gm5C...5’
?
?
180
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.18. CpG partially overlaps the recognition site.
?
?
Conventional FastDigest® Sequence* Effect
HincII HincII
5’...GTYRAm5C G...3’
3’...CARYT Gm5C...5’
5’...m5C GTYRAm5C G...3’
3’... Gm5CARYT Gm5C...5’
5’...GTm5C GAC...3’
3’...CA Gm5CTG...5’
HinfI HinfI
5’...GANTm5C G...3’
3’...CTNA Gm5C...5’
5’...m5C GANTm5C G...3’
3’... Gm5CTNA Gm5C...5’
HphI5’...m5C GGTGA...3’
3’... Gm5CCACT...5’
Hpy8I Hpy8I
5’...GTNNAm5C G...3’
3’...CANNT Gm5C...5’
5’...m5C GTNNAm5C G...3’
3’... Gm5CANNT Gm5C...5’
HpyF10VI HpyF10VI
5’...GC(N)7Gm5C G...3’
3’...CG(N)7C Gm5C...5’
5’...m5C GC(N)7Gm5C G...3’
3’... Gm5CG(N)7C Gm5C...5’
5’...Gm5C G(N)6GC...3’
3’...C Gm5C(N)6CG...5’
5’...Gm5C G(N)5m5C GC...3’
3’...C Gm5C(N)5 Gm5CG...5’
KpnI KpnI
5’...m5C GGTACm5C G...3’
3’... Gm5CCATG Gm5C...5’
KspAI HpaI
5’...GTTAAm5C G...3’
3’...CAATT Gm5C...5’
5’...m5C GTTAAm5C G...3’
3’... Gm5CAATT Gm5C...5’
LguI SapI
5’...m5C GCTCTTC...3’
3’... Gm5CGAGAAG...5’
5’...GCTCTTm5C G...3’
3’...CGAGAA Gm5C...5’
Lsp1109I BbvI
5’...m5C GCAGm5C G...3’
3’... Gm5CGTC Gm5C...5’
LweI SfaNI
5’...GCATm5C G...3’
3’...CGTA Gm5C...5’
MbiI BsrBI
5’...m5C GAGCGG...3’
3’... Gm5CTCGCC...5’
MboI MboI
5’...m5C GATm5C G...3’
3’... Gm5CTA Gm5C...5’
MboII MboII
5’...m5C GAAGA...3’
3’... Gm5CTTCT...5’
MnlI MnlI
5’...CCTm5C G...3’
3’...GGA Gm5C...5’
MssI MssI
5’...GTTTAAAm5C G...3’
3’...CAAATTT Gm5C...5’
5’...m5C GTTTAAAm5C G...3’
3’... Gm5CAAATTT Gm5C...5’
Mva1269I Mva1269I
5’...GAATGm5C G...3’
3’...CTTAC Gm5C...5’
5’...m5C GAATGC...3’
3’... Gm5CTTACG...5’
NheI NheI
5’...GCTAGm5C G...3’
3’...CGATC Gm5C...5’
5’...m5C GCTAGm5C G...3’
3’... Gm5CGATC Gm5C...5’
?
Conventional FastDigest® Sequence* Effect
SfiI SfiI
5’...m5C GGCC(N)5GGCm5C G...3’
3’... Gm5CCGG(N)5CCG Gm5C...5’
5’...GGCm5C G(N)4GGCC...3’
3’...CCG Gm5C(N)4CCGG...5’
5’...GGCm5C G(N)3m5C GGCC...3’
3’...CCG Gm5C(N)3 Gm5CCGG...5’
SgeI**
5’...m5C GNG...3’
3’... Gm5CNC...5’
5’...m5C Gm5C G...3’
3’... Gm5C Gm5C...5’
SmoI5’...CTm5C GAG...3’
3’...GA Gm5CTC...5’
TaaI TaaI
5’...Am5C GGT...3’
3’...T Gm5CCA...5’
TstI
5’...CAm5C G(N)5TCC...3’
3’...GT Gm5C(N)5AGG...5’
5’...CAC(N)5TCm5C G...3’
3’...GTG(N)5AG Gm5C...5’
XapI XapI
5’...m5C GAATTm5C G...3’
3’... Gm5CTTAA Gm5C...5’
XceI NspI
5’...RCATGm5C G...3’
3’...YGTAC Gm5C...5’
5’...m5C GCATGm5C G...3’
3’... Gm5CGTAC Gm5C...5’
XmiI AccI
5’...GTm5C GAC...3’
3’...CA Gm5CTG...5’
5’...GTMKAm5C G...3’
3’...CAKMT Gm5C...5’
Note* – recognition sequence is indicated in bold.
** – SgeI cleaves only methylated DNA.
– overlapping methylase sequences. m5C = 5-methylcytosine.
– cleavage not blocked.
– cleavage blocked.
–cleavage rate is reduced significantly by methylation.
?– the sensitivity to methylation has not been determined.
Single letter codeR = G or A; H = A, C or T; Y = C or T; V = A, C or G;W = A or T; B = C, G or T;M = A or C; D = A, G or T;K = G or T; N = G, A, T or C.S = C or G;
Conventional FastDigest® Sequence* Effect
NmuCI NmuCI
5’...GTCAm5C G...3’
3’...CAGT Gm5C...5’
5’...m5C GTCAm5C G...3’
3’... Gm5CAGT Gm5C...5’
OliI AleI
5’...CAm5C GNNNGTG...3’
3’...GT Gm5CNNNCAC...5’
5’...CAm5C GNNm5C GTG...3’
3’...GT Gm5CNN Gm5CAC...5’
PaeI SphI
5’...m5C GCATGm5C G...3’
3’... Gm5CGTAC Gm5C...5’
PdmI PdmI
5’...GAA(N)4TTm5C G...3’
3’...CTT(N)4AA Gm5C...5’
5’...m5C GAA(N)4TTm5C G...3’
3’... Gm5CTT(N)4AA Gm5C...5’
PfeI TfiI
5’...GAWTm5C G...3’
3’...CTWA Gm5C...5’
PfoI PfoI
5’...TCm5C GGGA...3’
3’...AG Gm5CCCT...5’
PpiI
5’...m5C GAAC(N)5CTC...3’
3’... Gm5CTTG(N)5GAG...5’
5’...GAAC(N)5CTm5C G...3’
3’...CTTG(N)5GA Gm5C...5’
5’...GAAm5C G(N)4CTC...3’
3’...CTT Gm5C(N)4GAG...5’
PspFI5’...CCCAGm5C G...3’
3’...GGGTC Gm5C...5’
PsuI PsuI
5’...m5C GGATCm5C G...3’
3’... Gm5CCTAG Gm5C...5’
PsyI PsyI
5’...GAm5C GNNGTm5C G...3’
3’...CT Gm5CNNCA Gm5C...5’
RsaI RsaI
5’...GTAm5C G...3’
3’...CAT Gm5C...5’
5’...m5C GTAm5C G...3’
3’... Gm5CAT Gm5C...5’
RseI MslI
5’...CAm5C GNNNRTG...3’
3’...GT Gm5CNNNYAC...5’
SacI SacI
5’...m5C GAGCTm5C G...3’
3’... Gm5CTCGA Gm5C...5’
SanDI
5’...GGGWCCm5C G...3’
3’...CCCWGG Gm5C...5’
5’...m5C GGGWCCm5C G...3’
3’... Gm5CCWGG Gm5C...5’
SatI Fnu4HI
5’...Gm5C GGC...3’
3’...C Gm5CCG...5’
5’...GCNGm5C G...3’
3’...CGNC Gm5C...5’
SchI MlyI
5’...GAGTm5C G...3’
3’...CTCA Gm5C...5’
5’...m5C GAGTC...3’
3’... Gm5CTCAG...5’
SduIBsp1286I
5’...m5C GDGCHm5C G...3’
3’... Gm5CHCGD Gm5C...5’
SfaNI
5’...m5C GCATC...3’
3’... Gm5CGTAG...5’
5’...GCATm5C G...3’
3’...CGTA Gm5C...5’
181
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Effect of EcoKI and EcoBI Methylation on DNA Cleavage
Methylated DNA substrates were purified from E.coli K12 or E.coli B strains.
Table 1.19. EcoBI overlapping methylation.
Conventional FastDigest® Sequence* Effect
AatIIAatII
5’...TGm6ACGTC(N)4 TGCT...3’
3’...AC TGCAG(N)4m6ACGA...5’
AluIAluI
5’...TGm6AGCT(N)5 TGCT...3’
3’...AC TCGA(N)5m6ACGA...5’
BclI BclI
5’...TGm6A TCA(N)5 TGCT...3’
3’...AC Tm6AGT(N)5m6ACGA...5’
BglII BglII
5’...TGm6AGATCT(N)3 TGCT...3’
3’...AC TCTAGA(N)3m6ACGA...5’
Bpu10IBpuI
5’...CCTGm6AGC(N)6 TGCT...3’
3’...GGAC TCG(N)6m6ACGA...5’
Bsp143ISau3AI
5’...TGm6ATC(N)6 TGCT...3’
3’...AC TAG(N)6m6ACGA...5’
EcoRI EcoRI
5’...TGm6AATTC(N)4 TGCT...3’
3’...AC TTAAG(N)4m6ACGA...5’
HincII HincII
5’...GTTGm6AC(N)7 TGCT...3’
3’...CAAC TG(N)7m6ACGA...5’
HindIII HindIII
5’...TGm6AAGCTT(N)3 TGCT...3’
3’...AC TTCGAA(N)3m6ACGA...5’
HinfI HinfI
5’...TGm6ANTC(N)5 TGCT...3’
3’...AC TNAG(N)5m6ACGA...5’
HphI 5’...GGTGm6A(N)8 TGCT...3’
3’...CCAC T(N)8m6ACGA...5’
MboI MboI
5’...TGm6ATC(N)6 TGCT...3’
3’...AC TAG(N)6m6ACGA...5’
MboII MboII
5’...TGm6AAGA(N)5 TGCT...3’
3’...AC TTCT(N)5m6ACGA...5’
MluI MluI
5’...TGm6ACGCGT(N)3 TGCT...3’
3’...AC TGCGCA(N)3m6ACGA...5’
MnlI MnlI
5’...TGm6AGG(N)6 TGCT...3’
3’...AC TCC(N)6m6ACGA...5’
NdeI NdeI
5’...TGm6A(N)4CATA TGCT...3’
3’...AC T(N)4GTATm6ACGA...5’
OliI AleI
5’...TGm6A(N)4CACG TGCT...3’
3’...AC T(N)4GTGCm6ACGA...5’
PaeI SphI
5’...TGm6A(N)5GCA TGCT...3’
3’...AC T(N)5CGTm6ACGA...5’
PagIBspHI
5’...TCATGm6A(N)8 TGCT...3’
3’...AGTAC T(N)8m6ACGA...5’
SacI SacI
5’...TGm6AGCTC(N)4 TGCT...3’
3’...AC TCGAG(N)4m6ACGA...5’
ScaI ScaI
5’...TGm6AGTACT(N)3 TGCT...3’
3’...AC TCATGA(N)3m6ACGA...5’
SspI SspI
5’...TGm6AATATT(N)3 TGCT...3’
3’...AC TTATAA(N)3m6ACGA...5’
TasI Tsp509I
5’...TGm6AATT(N)5 TGCT...3’
3’...AC TTAA(N)5m6ACGA...5’
Recognition sequences of the following endonu-cleases may also overlap with DNA sequences methylated by EcoBI and EcoKI.
The following conventional restriction enzymes and the respective FastDigest® enzymes have not been tested for sensitivity to EcoBI methylation: AarI, AdeI, AjiI, Alw21I, BauI, BcuI, BfiI, BglII, BoxI, BpiI, Bpu1102I, BseGI, BseJI, BseMI, BseNI, BseXI, BshTI, Bsp143II, BspPI, Bsu15I, BveI, CaiI, Cfr10I, CseI, DpnI, Eam1104I, Ecl136II, Eco24I, Eco31I, Eco32I, Eco47III, Eco57I, Eco57MI, Eco72I, Eco81I, Eco91I, Eco147I, EcoO109I, Esp3I, FspAI, FokI*, HaeII*, HindIII, Hin1I, Hin1II, Hin4I, Hpy8I, HpyF3I, LguI, Lsp1109I, LweI, MbiI, Mph1103I, MunI, Mva1269I, NdeI, NmuCI, PdmI, PfeI, Ppu21I, PscI, Psp5II, Psp1406I, PsuI, PsyI, PvuII, RseI, SchI, SduI, SexAI*, SfaNI*, SmiI, SmoI, SspI, TaaI, TaiI, TatI, TscAI, TstI, VspI, XagI, XapI and XceI.
The following conventional restriction enzymes and the respective FastDigest® enzymes have not been tested for sensitivity to EcoKI methylation: AanI, AdeI, AjiI, BauI, BcuI, BfiI, BseNI, BshNI, BshTI, Bsp119I, BveI, Cfr10I, Eco72I, Eco91I, FspAI, Hpy8I, MseI*, PacI, PdmI, Ppu21I, PscI, Psp1406I, SexAI*, SduI, TaaI, TaiI, TscAI, TsoI and XceI. * enzymes are available in FastDigest® format only.
Note* – recognition sequence is indicated in bold.
– cleavage not blocked.
– cleavage blocked.
–cleavage rate is reduced significantly by methylation.
Single letter codeR = G or A; H = A, C or T; Y = C or T; V = A, C or G;W = A or T; B = C, G or T;M = A or C; D = A, G or T;K = G or T; N = G, A, T or C.S = C or G;
Table 1.20. EcoKI overlapping methylation.
Conventional FastDigest® Sequence* Effect
Alw21I Alw21I
5’...Am6AC(N)6G TGCWC...3’
3’...T TG(N)6Cm6ACGWG...5’
Alw44I ApaLI
5’...Am6AC(N)6G TGCAC...3’
3’...T TG(N)6Cm6ACGTG...5’
BseSI Bme1580I
5’...Am6AC(N)6G TGCMC...3’
3’...T TG(N)6Cm6ACGKG...5’
DraI DraI
5’...TTTAAm6AC(N)6G TGC...3’
3’...AAATT TG(N)6Cm6ACG...5’
HincII HincII
5’...GTYAm6AC(N)6G TGC...3’
3’...CART TG(N)6Cm6ACG...5’
KspAI HpaI
5’...GTTAm6AC(N)6G TGC...3’
3’...CAAT TG(N)6Cm6ACG...5’
MluI MluI
5’...Am6ACGCGTNNG TGC...3’
3’...T TGCGCANNCm6ACG...5’
MssI MssI
5’...GTTTAAm6AC(N)6G TGC...3’
3’...CAAATT TG(N)6Cm6ACG...5’
NsbI FspI
5’...Am6AC(N)6G TGCGCA...3’
3’...T TG(N)6Cm6ACGCGT...5’
OliI AleI
5’...Am6ACAC(N)4G TGC...3’
3’...T TGTG(N)4Cm6ACG...5’
RseI MslI
5’...GCm6ACNNNNRTG TT...3’
3’...CG TGNNNNYACm6AT...5’
Tru1I Tru1I
5’...TTAm6AC(N)6G TGC...3’
3’...AAT TG(N)6Cm6ACG...5’
182
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Newly Generated Cleavage Sites
Recognition Sites Resulting from Ligation of Blunt DNA Ends
Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzymeRestriction enzymes that cleave the newly generated recognition sequence
AanI (PsiI)/FastDi-gest® PsiI (AanI) (TTA↓TAA)
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC), FaiI* (YA↓TA), FaiI* (YA↓TG) FaiIDraI/FastDigest® DraI (TTT↓AAA), Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/FastDigest® HpaI (KspAI) (GTT↓AAC), MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC), RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT)
FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT) SetIEco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT) AluI/FastDigest® AluI, CviJI, SetIEheI (SfoI)/FastDigest® EheI (GGC↓GCC) CviJI
SspI/FastDigest® SspI (AAT↓ATT)FastDigest® MseI (SaqAI), TasI (Tsp509I)/FastDigest® Tsp509I (TasI), Tru1I (MseI)/FastDigest® Tru1I
AjiI (BmgBI)* (CAC↓GTC)
AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
SetI
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI, SetIEco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA)
MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI
Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG)
Eco72I (PmlI)/FastDigest® PmlI (Eco72I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI
FaiI* (YA↓TG) TscAI (TspRI)/FastDigest® TspRI (TscAI)MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIPdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC) BceAIZraI (GAC↓GTC) AjiI (BmgBI), MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI
AjiI (BmgBI)* (GAC↓GTG)
AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
SetI
DpnI/FastDigest® DpnI (GA↓TC) HinfI/FastDigest® HinfIEcl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI, SetIEco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG)
MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI
Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA)
CseI (HgaI)/FastDigest® HgaI (CseI)
EheI (SfoI)/FastDigest® EheI (GGC↓GCC)CseI (HgaI)/FastDigest® HgaI (CseI), Hin1I (BsaHI)/FastDi-gest® BsaHI (Hin1I)
MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIPdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC) BceAI
ZraI (GAC↓GTC)AatII/FastDigest® AatII, Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I), MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI, ZraI
AluI/FastDigest® AluI (AG↓CT)
AjiI (BmgBI)* (CAC↓GTC), AjiI (BmgBI)* (GAC↓GTG), Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG), ZraI (GAC↓GTC)
SetI
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaIBsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)
CviJI
CviJI* (RG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
AluI/FastDigest® AluI, CviJI, SetI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
EheI (SfoI)/FastDigest® EheI (GGC↓GCC) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI
Bsh1236I (BstUI)/FastDigest® Bsh1236I (CG↓CG)
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT) CviJIEheI (SfoI)/FastDigest® EheI (GGC↓GCC) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJIBsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG)
Bsh1236I (BstUI)/FastDigest® Bsh1236I
Single letter codeR = G or A; H = A, C or T; Y = C or T; V = A, C or G;W = A or T; B = C, G or T;M = A or C; D = A, G or T;K = G or T; N = G, A, T or C.S = C or G;
Note* Restriction enzymes that have degenerate recognition
sequences (i.e., recognize more than one sequence). Be aware that these restriction endonucleases will cleave sequences in addition to the one listed.
• Fermentas FastDigest® enzymes are shown in ruby.• Fermentas conventional enzymes are shown in orange.
183
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzymeRestriction enzymes that cleave the newly generated recognition sequence
Bsp68I (NruI)/Fast-Digest® NruI (RruI) (TCG↓CGA)
Bsh1236I (BstUI)/FastDigest® Bsh1236I (CG↓CG), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG)
Bsh1236I (BstUI)/FastDigest® Bsh1236I
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaIDraI/FastDigest® DraI (TTT↓AAA), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/Fast-Digest® HpaI (KspAI) (GTT↓AAC), MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC), RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT), SspI/FastDigest® SspI (AAT↓ATT)
TaqI/FastDigest® TaqI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, TaqI/Fast-Digest® TaqI
Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT) CviJIEheI (SfoI)/FastDigest® EheI (GGC↓GCC) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJIHincII (HindII)/FastDigest® HincII* (GTY↓GAC) Hpy188I
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC)
AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), FaiI* (YA↓TA), FaiI* (YA↓TG) FaiIAluI/FastDigest® AluI (AG↓CT), Bsh1236I (BstUI)/FastDigest® Bsh1236I (CG↓CG), Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), CviJI* (RG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), HpyCH4V (TG↓CA), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA), MspA1I* (CMG↓CGG), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI
Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT)Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI, SetI
Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT) AluI/FastDigest® AluI, CviJI, SetIEheI (SfoI)/FastDigest® EheI (GGC↓GCC) CviJIHincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/FastDigest® HpaI (KspAI) (GTT↓AAC) Hpy8I (MjaIV)/FastDigest® Hpy8I
HincII (HindII)/FastDigest® HincII* (GTY↓GAC)Hpy8I (MjaIV)/FastDigest® Hpy8I, XmiI (AccI)/FastDigest® AccI (XmiI)
RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT) MaeIIISspI/FastDigest® SspI (AAT↓ATT) TasI (Tsp509I)/FastDigest® Tsp509I (TasI)
BsuRI (HaeIII)/FastDi-gest® HaeIII (BsuRI) (GG↓CC)
AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
CviJI
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaICviJI* (RG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)
BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
EheI (SfoI)/FastDigest® EheI (GGC↓GCC)BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI
FspAI/FastDigest® FspAI* (RTGC↓GCAC)BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
HincII (HindII)/FastDigest® HincII* (GTY↓GAC) FaqI (BsmFI)/FastDigest® BsmFI (FaqI)
CviJI* (AG↓CY)
AjiI (BmgBI)* (CAC↓GTC), AjiI (BmgBI)* (GAC↓GTG), Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG), ZraI (GAC↓GTC)
SetI
AluI/FastDigest® AluI (AG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/Fast-Digest® BsrBI (MbiI)* (CCG↓CTC), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
AluI/FastDigest® AluI, CviJI, SetI
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaIBsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)
CviJI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
EheI (SfoI)/FastDigest® EheI (GGC↓GCC) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI
CviJI* (GG↓CY)
AluI/FastDigest® AluI (AG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
CviJI
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaIBsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)
BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
EheI (SfoI)/FastDigest® EheI (GGC↓GCC)BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI
FspAI/FastDigest® FspAI* (RTGC↓GCAC)BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
HincII (HindII)/FastDigest® HincII* (GTY↓GAC) FaqI (BsmFI)/FastDigest® BsmFI (FaqI)
184
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzymeRestriction enzymes that cleave the newly generated recognition sequence
DpnI/FastDigest® DpnI (GA↓TC)
AjiI (BmgBI)* (CAC↓GTC), ZraI (GAC↓GTC)HinfI/FastDigest® HinfI, PleI, SchI (MlyI)/FastDigest® MlyI (SchI)
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) HinfI/FastDigest® HinfIEco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT) SetIEco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC) HinfI/FastDigest® HinfI, PfeI (TfiI)/FastDigest® TfiI (PfeI)Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT) AluI/FastDigest® AluI, CviJI, SetIEheI (SfoI)/FastDigest® EheI (GGC↓GCC) CviJI
FspAI/FastDigest® FspAI* (RTGC↓GCAC)Alw21I (BsiHKAI)/FastDigest® Alw21I, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
SspI/FastDigest® SspI (AAT↓ATT) TasI (Tsp509I)/FastDigest® Tsp509I (TasI)
DraI/FastDigest® DraI (TTT↓AAA)
AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/FastDigest® HpaI (KspAI) (GTT↓AAC)
FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) TaqI/FastDigest® TaqIFspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDi-gest® FspI (NsbI) (TGC↓GCA)
HpyCH4V
MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT)DraI/FastDigest® DraI, FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC)
AjiI (BmgBI)* (CAC↓GTC), AjiI (BmgBI)* (GAC↓GTG), Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG), ZraI (GAC↓GTC)
SetI
AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
AluI/FastDigest® AluI, CviJI, SetI
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaIBsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)
CviJI
DpnI/FastDigest® DpnI (GA↓TC)HinfI/FastDigest® HinfI, PleI, SchI (MlyI)/FastDigest® MlyI (SchI)
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
EheI (SfoI)/FastDigest® EheI (GGC↓GCC) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI
MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC)
AluI/FastDigest® AluI, Alw21I (BsiHKAI)/FastDigest® Alw21I, CviJI, Ecl136II (EcoICRI)/FastDigest® Ecl136II, Eco24I (BanII), SacI/FastDigest® SacI, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI), SetI
Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA)
AjiI (BmgBI)* (CAC↓GTC), ZraI (GAC↓GTC) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiIAjiI (BmgBI)* (GAC↓GTG), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), Ppu21I (BsaAI)/FastDi-gest® BsaAI (Ppu21I)* (YAC↓GTG)
MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI
AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
SetI
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI, SetIMbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIPdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC) BceAI
Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA)Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI
Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT)
AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), DpnI/FastDigest® DpnI (GA↓TC), FaiI* (YA↓TA), FaiI* (YA↓TG)
SetI
AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
CviJI
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC)Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI, SetI
BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)
BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
EheI (SfoI)/FastDigest® EheI (GGC↓GCC)BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI
FspAI/FastDigest® FspAI* (RTGC↓GCAC)BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
HincII (HindII)/FastDigest® HincII* (GTY↓GAC) FaqI (BsmFI)/FastDigest® BsmFI (FaqI)
185
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzymeRestriction enzymes that cleave the newly generated recognition sequence
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)
AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA) FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1IAluI/FastDigest® AluI (AG↓CT), Bsh1236I (BstUI)/FastDigest® Bsh1236I (CG↓CG), BsuRI (HaeIII)/Fast-Digest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), CviJI* (RG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), HpyCH4V (TG↓CA), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA), MspA1I* (CMG↓CGG), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, TaqI/Fast-Digest® TaqI
DpnI/FastDigest® DpnI (GA↓TC) HinfI/FastDigest® HinfI, PfeI (TfiI)/FastDigest® TfiI (PfeI)FspAI/FastDigest® FspAI* (RTGC↓GCAC), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA) HpyCH4VFspAI/FastDigest® FspAI* (RTGC↓GCAT) HpyCH4V, Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I)
Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT)
AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC), DpnI/FastDigest® DpnI (GA↓TC), FaiI* (YA↓TA), FaiI* (YA↓TG)
AluI/FastDigest® AluI, CviJI, SetI
AluI/FastDigest® AluI (AG↓CT), Bsh1236I (BstUI)/FastDigest® Bsh1236I (CG↓CG), Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), CviJI* (RG↓CT), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), HpyCH4V (TG↓CA), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
CviJI
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) CviJI, MnlI/FastDigest® MnlIEco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC) LweI (SfaNI)/FastDigest® SfaNI (BmsI)
EheI (SfoI)/FastDigest® EheI (GGC↓GCC)FastDigest® HaeII (BfoI), HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDi-gest® FspI (NsbI) (TGC↓GCA)
HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG)CviJI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
PdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC)SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), TauI/FastDi-gest® TauI
SrfI (GCCC↓GGGC) Cac8I
Eco72I (PmlI)/FastDi-gest® PmlI (Eco72I) (CAC↓GTG)
AjiI (BmgBI)* (CAC↓GTC), ZraI (GAC↓GTC) AjiI (BmgBI), MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI
AjiI (BmgBI)* (GAC↓GTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG)Eco72I (PmlI)/FastDigest® PmlI (Eco72I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI
AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
SetI
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI, SetIEco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA)
MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI
FaiI* (YA↓TG) TscAI (TspRI)/FastDigest® TspRI (TscAI)MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIPdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC) BceAI
EheI (SfoI)/FastDigest® EheI (GGC↓GCC)
AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC), DpnI/FastDigest® DpnI (GA↓TC), FaiI* (YA↓TA), FaiI* (YA↓TG)
CviJI
AjiI (BmgBI)* (CAC↓GTC), ZraI (GAC↓GTC) Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)AluI/FastDigest® AluI (AG↓CT), Bsh1236I (BstUI)/FastDigest® Bsh1236I (CG↓CG), Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA), CviJI* (RG↓CT), HpyCH4V (TG↓CA), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI
BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)
BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC)BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, MnlI/FastDigest® MnlI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC) LweI (SfaNI)/FastDigest® SfaNI (BmsI)
Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT)FastDigest® HaeII (BfoI), HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDi-gest® FspI (NsbI) (TGC↓GCA)
HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG)BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
PdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC)SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), TauI/FastDi-gest® TauI
SrfI (GCCC↓GGGC) Cac8I
FaiI* (CA↓TR)
AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC)
FaiI
AjiI (BmgBI)* (GAC↓GTG), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), MspA1I* (CMG↓CTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG), PvuII/FastDigest® PvuII (CAG↓CTG)
TscAI (TspRI)/FastDigest® TspRI (TscAI)
Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT) SetIEco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT) AluI/FastDigest® AluI, CviJI, SetIEheI (SfoI)/FastDigest® EheI (GGC↓GCC) CviJISspI/FastDigest® SspI (AAT↓ATT) TasI (Tsp509I)/FastDigest® Tsp509I (TasI)
186
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzymeRestriction enzymes that cleave the newly generated recognition sequence
FaiI* (TA↓TR)
AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC)
FaiI
Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT) SetIEco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT) AluI/FastDigest® AluI, CviJI, SetIEheI (SfoI)/FastDigest® EheI (GGC↓GCC) CviJISspI/FastDigest® SspI (AAT↓ATT) TasI (Tsp509I)/FastDigest® Tsp509I (TasI)
FspAI/FastDigest® FspAI* (ATGC↓GCAY)
DraI/FastDigest® DraI (TTT↓AAA), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/Fast-Digest® HpaI (KspAI) (GTT↓AAC), MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC), RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT)
HpyCH4VI
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)LweI (SfaNI)/FastDigest® SfaNI (BmsI), HpyCH4V, Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I)
Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), EheI (SfoI)/FastDigest® EheI (GGC↓GCC)HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA)HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), NsbI (FspI)/FastDigest® FspI (NsbI)
PdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC)SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), TauI/FastDi-gest® TauI
SrfI (GCCC↓GGGC) Cac8ISspI/FastDigest® SspI (AAT↓ATT) HpyCH4V, Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I)
FspAI/FastDigest® FspAI* (GTGC↓GCAY)
BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)
BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
DpnI/FastDigest® DpnI (GA↓TC)Alw21I (BsiHKAI)/FastDigest® Alw21I, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
DraI/FastDigest® DraI (TTT↓AAA), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/FastDi-gest® HpaI (KspAI) (GTT↓AAC), MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT), SspI/FastDigest® SspI (AAT↓ATT)
HpyCH4V
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlIEco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC) LweI (SfaNI)/FastDigest® SfaNI (BmsI), HpyCH4V
Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), EheI (SfoI)/FastDigest® EheI (GGC↓GCC)HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA)HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), NsbI (FspI)/FastDigest® FspI (NsbI)
PdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC)SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), TauI/FastDi-gest® TauI
RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT)
Alw21I (BsiHKAI)/FastDigest® Alw21I, Alw44I (ApaLI)/FastDigest® ApaLI (AIw44I), BseSI (Bme1580I)/Fast-Digest® Bme1580I (BseSI), Hpy8I (MjaIV)/FastDigest® Hpy8I, HpyCH4V, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
SrfI (GCCC↓GGGC) Cac8I
HincII (HindII)/FastDigest® HincII* (GTC↓RAC)
Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) Hpy188I
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC)Hpy8I (MjaIV)/FastDigest® Hpy8I, XmiI (AccI)/FastDigest® AccI (XmiI)
DpnI/FastDigest® DpnI (GA↓TC) Alw26I (BsmAI)/FastDigest® Alw26IEcl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI
KspAI (HpaI)/FastDigest® HpaI (KspAI) (GTT↓AAC)HincII (HindII)/FastDigest® HincII, Hpy8I (MjaIV)/FastDi-gest® Hpy8I
MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIRsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT) MaeIII, NmuCI (Tsp45I)/FastDigest® NmuCI
HincII (HindII)/FastDigest® HincII* (GTT↓RAC)
AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA), DraI/FastDigest® DraI (TTT↓AAA), MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT)
FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) TaqI/FastDigest® TaqIBst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Hpy8I (MjaIV)/FastDigest® Hpy8IFspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDi-gest® FspI (NsbI) (TGC↓GCA)
HpyCH4V
KspAI (HpaI)/FastDigest® HpaI (KspAI) (GTT↓AAC)HincII (HindII)/FastDigest® HincII, Hpy8I (MjaIV)/Fast-Digest® Hpy8I, KspAI (HpaI)/FastDigest® HpaI (KspAI), FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT) MaeIII
HpyCH4V (TG↓CA)
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT) CviJIEheI (SfoI)/FastDigest® EheI (GGC↓GCC) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI
187
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzymeRestriction enzymes that cleave the newly generated recognition sequence
KspAI (HpaI)/FastDi-gest® HpaI (KspAI) (GTT↓AAC)
AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), DraI/FastDigest® DraI (TTT↓AAA), MssI (PmeI)/FastDi-gest® MssI (GTTT↓AAAC), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT)
FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) TaqI/FastDigest® TaqIBst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Hpy8I (MjaIV)/FastDigest® Hpy8IFspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDi-gest® FspI (NsbI) (TGC↓GCA)
HpyCH4V
HincII (HindII)/FastDigest® HincII* (GTY↓AAC)HincII (HindII)/FastDigest® HincII, Hpy8I (MjaIV)/Fast-Digest® Hpy8I, KspAI (HpaI)/FastDigest® HpaI (KspAI), FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
HincII (HindII)/FastDigest® HincII* (GTY↓GAC)HincII (HindII)/FastDigest® HincII, Hpy8I (MjaIV)/FastDi-gest® Hpy8I
RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT) MaeIII
MbiI (BsrBI)/FastDi-gest® BsrBI (MbiI)* (CCG↓CTC)
AjiI (BmgBI)* (CAC↓GTC), AjiI (BmgBI)* (GAC↓GTG), Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), HincII (HindII)/FastDigest® HincII* (GTY↓GAC), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG), ZraI (GAC↓GTC)
HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
AluI/FastDigest® AluI (AG↓CT), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), CviJI* (RG↓CT), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), HpyCH4V (TG↓CA), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)
SsiI (AciI)/FastDigest® AciI (SsiI)
Bsh1236I (BstUI)/FastDigest® Bsh1236I (CG↓CG), Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA)Bsh1236I (BstUI)/FastDigest® Bsh1236I, SsiI (AciI)/FastDigest® AciI (SsiI)
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC)MbiI (BsrBI)/FastDigest® BsrBI (MbiI), SsiI (AciI)/FastDi-gest® AciI (SsiI)
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT)CviJI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
EheI (SfoI)/FastDigest® EheI (GGC↓GCC)BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
MspA1I* (CMG↓CGG)BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Bsh1236I (BstUI)/FastDigest® Bsh1236I, BtgI, Cfr42I (SacII), MspA1I, SsiI (AciI)/FastDigest® AciI (SsiI)
MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG) MspA1I, SsiI (AciI)/FastDigest® AciI (SsiI)
PdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC)BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
SmaI/FastDigest® SmaI (CCC↓GGG), SrfI (GCCC↓GGGC)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BseDI (BsaJI)/FastDigest® BsaJI (BseDI), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
MbiI (BsrBI)/FastDi-gest® BsrBI (MbiI)* (GAG↓CGG)
AjiI (BmgBI)* (CAC↓GTC), AjiI (BmgBI)* (GAC↓GTG), Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG), ZraI (GAC↓GTC)
SetI
AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
AluI/FastDigest® AluI, CviJI, SetI
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaIBsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)
CviJI
DpnI/FastDigest® DpnI (GA↓TC)HinfI/FastDigest® HinfI, PleI, SchI (MlyI)/FastDigest® MlyI (SchI)
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC)
AluI/FastDigest® AluI, Alw21I (BsiHKAI)/FastDigest® Alw21I, CviJI, Ecl136II (EcoICRI)/FastDigest® Ecl136II, Eco24I (BanII), SacI/FastDigest® SacI, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI), SetI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
EheI (SfoI)/FastDigest® EheI (GGC↓GCC) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI
188
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzymeRestriction enzymes that cleave the newly generated recognition sequence
MlsI (MscI)/FastDi-gest® MscI (MlsI) (TGG↓CCA)
AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
CviJI
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaIBsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT)
BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
EheI (SfoI)/FastDigest® EheI (GGC↓GCC)BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI
FspAI/FastDigest® FspAI* (RTGC↓GCAC)BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
HincII (HindII)/FastDigest® HincII* (GTY↓GAC) FaqI (BsmFI)/FastDigest® BsmFI (FaqI)
MspA1I* (CAG↓CKG)
AjiI (BmgBI)* (CAC↓GTC), AjiI (BmgBI)* (GAC↓GTG), Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG), ZraI (GAC↓GTC)
SetI
AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC)
AluI/FastDigest® AluI, CviJI, SetI
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaIBsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)
CviJI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
EheI (SfoI)/FastDigest® EheI (GGC↓GCC) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJIFaiI* (YA↓TG) TscAI (TspRI)/FastDigest® TspRI (TscAI)MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG) MspA1I
PvuII/FastDigest® PvuII (CAG↓CTG)AluI/FastDigest® AluI, CviJI, MspA1I, PvuII/FastDigest® PvuII, SetI
MspA1I* (CCG↓CKG)
AjiI (BmgBI)* (CAC↓GTC), AjiI (BmgBI)* (GAC↓GTG), Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), HincII (HindII)/FastDigest® HincII* (GTY↓GAC), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG), ZraI (GAC↓GTC)
HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
AluI/FastDigest® AluI (AG↓CT), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), CviJI* (RG↓CT), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), HpyCH4V (TG↓CA), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)
SsiI (AciI)/FastDigest® AciI (SsiI)
Bsh1236I (BstUI)/FastDigest® Bsh1236I (CG↓CG),Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA)Bsh1236I (BstUI)/FastDigest® Bsh1236I, SsiI (AciI)/FastDigest® AciI (SsiI)
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC)MbiI (BsrBI)/FastDigest® BsrBI (MbiI), SsiI (AciI)/FastDi-gest® AciI (SsiI)
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT)CviJI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
EheI (SfoI)/FastDigest® EheI (GGC↓GCC)BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG)BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Bsh1236I (BstUI)/FastDigest® Bsh1236I, BtgI, Cfr42I (SacII), MspA1I, SsiI (AciI)/FastDigest® AciI (SsiI)
PdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC)BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
PvuII/FastDigest® PvuII (CAG↓CTG) MspA1I, SsiI (AciI)/FastDigest® AciI (SsiI)
SmaI/FastDigest® SmaI (CCC↓GGG), SrfI (GCCC↓GGGC)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BseDI (BsaJI)/FastDigest® BsaJI (BseDI), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC)
AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/FastDigest® HpaI (KspAI) (GTT↓AAC)
FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I,
Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) TaqI/FastDigest® TaqI
DraI/FastDigest® DraI (TTT↓AAA), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT)DraI/FastDigest® DraI, FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDi-gest® FspI (NsbI) (TGC↓GCA)
HpyCH4V
RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT) Hpy8I (MjaIV)/FastDigest® Hpy8I
189
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzymeRestriction enzymes that cleave the newly generated recognition sequence
NsbI (FspI)/FastDi-gest® FspI (NsbI) (TGC↓GCA)
DraI/FastDigest® DraI (TTT↓AAA), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/Fast-Digest® HpaI (KspAI) (GTT↓AAC), MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC), RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT), SspI/FastDigest® SspI (AAT↓ATT)
HpyCH4V
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlIEco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC) LweI (SfaNI)/FastDigest® SfaNI (BmsI) , HpyCH4V
Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), EheI (SfoI)/FastDigest® EheI (GGC↓GCC)HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT)HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), NsbI (FspI)/FastDigest® FspI (NsbI)
MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
PdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC)SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), TauI/FastDi-gest® TauI
SrfI (GCCC↓GGGC) Cac8I
PdiI (NaeI)/FastDi-gest® NaeI (PdiI) (GCC↓GGC)
DpnI/FastDigest® DpnI (GA↓TC), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC)
MnlI/FastDigest® MnlI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC) BccIEco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), EheI (SfoI)/FastDigest® EheI (GGC↓GCC), FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA)
SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), SsiI (AciI)/FastDi-gest® AciI (SsiI), TauI/FastDigest® TauI
MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG), SmaI/FastDigest® SmaI (CCC↓GGG), SrfI (GCCC↓GGGC)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (CAC↓GTR)
AjiI (BmgBI)* (CAC↓GTC), ZraI (GAC↓GTC) AjiI (BmgBI), MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI
AjiI (BmgBI)* (GAC↓GTG), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG)Eco72I (PmlI)/FastDigest® PmlI (Eco72I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI
AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
SetI
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI, SetI
Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA)MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI
FaiI* (YA↓TG) TscAI (TspRI)/FastDigest® TspRI (TscAI)MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIPdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC) BceAI
Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (TAC↓GTR)
AjiI (BmgBI)* (CAC↓GTC), ZraI (GAC↓GTC) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI
AjiI (BmgBI)* (GAC↓GTG), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG)MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI
AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
SetI
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI, SetI
Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA)Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI
MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIPdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC) BceAI
PvuII/FastDigest® PvuII (CAG↓CTG)
AjiI (BmgBI)* (CAC↓GTC), AjiI (BmgBI)* (GAC↓GTG), Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG), ZraI (GAC↓GTC)
SetI
AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC)
AluI/FastDigest® AluI, CviJI, SetI
Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaIBsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)
CviJI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
EheI (SfoI)/FastDigest® EheI (GGC↓GCC) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJIFaiI* (YA↓TG) TscAI (TspRI)/FastDigest® TspRI (TscAI)MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) MspA1I
MspA1I* (CMG↓CTG)AluI/FastDigest® AluI, CviJI, MspA1I, PvuII/FastDigest® PvuII, SetI
190
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzymeRestriction enzymes that cleave the newly generated recognition sequence
RsaI/FastDigest® RsaI (GT↓AC)
AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA) FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1IBsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) TaqI/FastDigest® TaqIBst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/FastDigest® HpaI (KspAI) (GTT↓AAC)
MaeIII
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) Alw26I (BsmAI)/FastDigest® Alw26I
FspAI/FastDigest® FspAI* (RTGC↓GCAC)
Alw21I (BsiHKAI)/FastDigest® Alw21I, Alw44I (ApaLI)/FastDigest® ApaLI (Alw44I), BseSI (Bme1580I)/Fast-Digest® Bme1580I (BseSI), Hpy8I (MjaIV)/FastDigest® Hpy8I, HpyCH4V, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA) HpyCH4VHincII (HindII)/FastDigest® HincII* (GTY↓GAC) MaeIII, NmuCI (Tsp45I)/FastDigest® NmuCIMssI (PmeI)/FastDigest® MssI (GTTT↓AAAC) Hpy8I (MjaIV)/FastDigest® Hpy8IScaI/FastDigest® ScaI (AGT↓ACT) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI
ScaI/FastDigest® ScaI (AGT↓ACT)
AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1IBsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) TaqI/FastDigest® TaqIBst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/FastDigest® HpaI (KspAI) (GTT↓AAC)
MaeIII
Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) Alw26I (BsmAI)/FastDigest® Alw26I
FspAI/FastDigest® FspAI* (RTGC↓GCAC)
Alw21I (BsiHKAI)/FastDigest® Alw21I, Alw44I (ApaLI)/FastDigest® ApaLI (Alw44I), BseSI (Bme1580I)/Fast-Digest® Bme1580I (BseSI), Hpy8I (MjaIV)/FastDigest® Hpy8I, HpyCH4V, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA) HpyCH4VHincII (HindII)/FastDigest® HincII* (GTY↓GAC) MaeIII, NmuCI (Tsp45I)/FastDigest® NmuCIMssI (PmeI)/FastDigest® MssI (GTTT↓AAAC) Hpy8I (MjaIV)/FastDigest® Hpy8IRsaI/FastDigest® RsaI (GT↓AC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI
SmaI/FastDigest® SmaI (CCC↓GGG)
DpnI/FastDigest® DpnI (GA↓TC), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC)
MnlI/FastDigest® MnlI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC) BccIEco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), EheI (SfoI)/FastDigest® EheI (GGC↓GCC), FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA)
SmuI (FauI), SsiI (AciI)/FastDigest® AciI (SsiI)
MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BseDI (BsaJI)/FastDigest® BsaJI (BseDI), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
PdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC)BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
SrfI (GCCC↓GGGC)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BmeT110I, BseDI (BsaJI)/FastDigest® BsaJI (BseDI), BssKI, Cfr9I (XmaI), Eco88I (AvaI)/FastDigest® AvaI (Eco88I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, SmaI/FastDigest® SmaI
SmiI (SwaI)/FastDi-gest® SwaI (SmiI) (ATTT↓AAAT)
AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/FastDigest® HpaI (KspAI) (GTT↓AAC)
FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) TaqI/FastDigest® TaqI
DraI/FastDigest® DraI (TTT↓AAA), MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC)DraI/FastDigest® DraI, FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDi-gest® FspI (NsbI) (TGC↓GCA)
HpyCH4V
191
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends.
First restriction enzyme
Second restriction enzymeRestriction enzymes that cleave the newly generated recognition sequence
SrfI (GCCC↓GGGC)
DpnI/FastDigest® DpnI (GA↓TC), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC)
MnlI/FastDigest® MnlI
Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC) BccIEco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), EheI (SfoI)/FastDigest® EheI (GGC↓GCC), FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA)
Cac8I, SmuI (FauI), SsiI (AciI)/FastDigest® AciI (SsiI)
MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BseDI (BsaJI)/FastDigest® BsaJI (BseDI), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
PdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC)BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
SmaI/FastDigest® SmaI (CCC↓GGG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BmeT110I, BseDI (BsaJI)/FastDigest® BsaJI (BseDI), BssKI, Cfr9I (XmaI), Eco88I (AvaI)/FastDigest® AvaI (Eco88I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, SmaI/FastDigest® SmaI
SspI/FastDigest® SspI (AAT↓ATT)
AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA)FastDigest® MseI (SaqAI), TasI (Tsp509I)/FastDigest® Tsp509I (TasI), Tru1I (MseI)/FastDigest® Tru1I
Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) TaqI/FastDigest® TaqIBst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC), DpnI/FastDigest® DpnI (GA↓TC), FaiI* (YA↓TA), FaiI* (YA↓TG)
TasI (Tsp509I)/FastDigest® Tsp509I (TasI)
FspAI/FastDigest® FspAI* (RTGC↓GCAC), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA) HpyCH4VFspAI/FastDigest® FspAI* (RTGC↓GCAT) HpyCH4V, Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I)
ZraI (GAC↓GTC)
AjiI (BmgBI)* (CAC↓GTC)AatII/FastDigest® AatII, Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I), MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI, ZraI
AjiI (BmgBI)* (GAC↓GTG), Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG)
MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI
AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)
SetI
DpnI/FastDigest® DpnI (GA↓TC) HinfI/FastDigest® HinfIEcl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI, SetIEco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA)
CseI (HgaI)/FastDigest® HgaI (CseI)
EheI (SfoI)/FastDigest® EheI (GGC↓GCC)CseI (HgaI)/FastDigest® HgaI (CseI), Hin1I (BsaHI)/FastDi-gest® BsaHI (Hin1I)
MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIPdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC) BceAI
192
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Recognition Sites Resulting from Ligation of Protruding Compatible DNA Ends
Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzyme
Restriction enzyme that cleave the newly generated recognition sequence
AatII/FastDigest® AatII (GACGT↓C)
SetI* (ACGT), TaiI (MaeII)/FastDigest® TaiI (ACGT) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI
AbsI (CC↓TCGAGG)
Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓TCGAG), SmoI (SmlI)* (C↓TCGAG), XhoI/FastDigest® XhoI (C↓TCGAG)
BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), MnlI/FastDigest® MnlI, SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI
PspXI* (VC↓TCGAGC), PspXI* (VC↓TCGAGT)BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), MnlI/FastDigest® MnlI, PspXI, SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI
PspXI* (VC↓TCGAGG)AbsI, BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), MnlI/FastDigest® MnlI, PspXI, SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI
SalI/FastDigest® SalI (G↓TCGAC) MnlI/FastDigest® MnlI, TaqI/FastDigest® TaqISgrDI (CG↓TCGACG) Hpy99I, MnlI/FastDigest® MnlI, TaqI/FastDigest® TaqI
Acc65I (Asp718I)/FastDi-gest® Acc65I (G↓GTACC)
BshNI (BanI)/FastDigest® BanI (BshNI)* (G↓GTACC)Acc65I (Asp718I)/FastDigest® Acc65I, BshNI (BanI)/FastDigest® BanI (BshNI), BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Csp6I (CviQI)/FastDigest® Csp6I, KpnI/FastDigest® KpnI, RsaI/FastDigest® RsaI
Bsp1407I (BsrGI)/FastDigest® Bsp1407I (T↓GTACA), Pfl23II (BsiWI)/FastDi-gest® BsiWI (Pfl23II) (C↓GTACG), TatI/FastDigest® TatI* (W↓GTACA), TatI/FastDigest® TatI* (W↓GTACT)
Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI
AflIII* (A↓CATGT)
BtgI* (C↓CATGG), Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CATGG), FatI (↓CATG), NcoI/FastDigest® NcoI (C↓CATGG), PagI (BspHI)/FastDigest® BspHI (PagI) (T↓CATGA)
CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)
PscI (PciI) (A↓CATGT)AflIII, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), PscI (PciI), XceI (NspI)/FastDigest® NspI (XceI)
AflIII* (A↓CGCGT)
BtgI* (C↓CGCGG) Bsh1236I (BstUI)/FastDigest® Bsh1236IMauBI/FastDigest® MauBI (CG↓CGCGCG), SgsI (AscI)/FastDigest® AscI (SgsI) (GG↓CGCGCC), PauI (BssHII)/FastDigest® BssHII (PteI) (G↓CGCGC)
Bsh1236I (BstUI)/FastDigest® Bsh1236I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
MluI/FastDigest® MluI (A↓CGCGT) AflIII, Bsh1236I (BstUI)/FastDigest® Bsh1236I, MluI/FastDigest® MluIAflIII* (A↓CGTGT) BtgI* (C↓CGTGG) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiIAlw21I (BsiHKAI)/FastDi-gest® Alw21I* (GAGCA↓C)
SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GAGCA↓C)Alw21I (BsiHKAI)/FastDigest® Alw21I, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
Alw21I (BsiHKAI)/FastDi-gest® Alw21I* (GAGCT↓C)
Eco24I (BanII)* (GAGCT↓C), SacI/FastDigest® SacI (GAGCT↓C), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GAGCT↓C)
AluI/FastDigest® AluI, Alw21I (BsiHKAI)/FastDigest® Alw21I, CviJI, Ecl136II (EcoICRI)/FastDigest® Ecl136II, Eco24I (BanII), SacI/FastDigest® SacI, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI), SetI
SetI* (AGCT) AluI/FastDigest® AluI, CviJI, SetI
Alw21I (BsiHKAI)/FastDi-gest® Alw21I* (GTGCA↓C)
BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI)* (GTGCA↓C), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GTGCA↓C)
Alw21I (BsiHKAI)/FastDigest® Alw21I, Alw44I (ApaLI)/FastDigest® ApaLI (Alw44I), BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), Hpy8I (MjaIV)/FastDigest® Hpy8I, HpyCH4V, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I) (ATGCA↓T) HpyCH4VPstI/FastDigest® PstI (CTGCA↓G), SdaI (SbfI)/FastDigest® SbfI (SdaI) (CCTGCA↓GG)
BsgI, HpyCH4V
Alw21I (BsiHKAI)/FastDi-gest® Alw21I* (GTGCT↓C)
SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GTGCT↓C)Alw21I (BsiHKAI)/FastDigest® Alw21I, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
Alw44I (ApaLI)/FastDigest® ApaLI (Alw44I) (G↓TGCAC)
BfmI (SfcI)/FastDigest® SfcI (BfmI)* (C↓TGCAG) BsgI, HpyCH4V
ApaI/FastDigest® ApaI (GGGCC↓C)
BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI)* (GGGCC↓C), Eco24I (BanII)* (GGGCC↓C), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GGGCC↓C)
ApaI/FastDigest® ApaI, BmgT120I, BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), Bsp120I (PspOMI)/FastDigest® Bsp120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
BamHI/FastDigest® BamHI (G↓GATCC)
BclI/FastDigest® BclI (T↓GATCA), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I) (↓GATC), MboI/FastDigest® MboI (↓GATC)
Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDi-gest® DpnI, MboI/FastDigest® MboI
BglII/FastDigest® BglII (A↓GATCT), PsuI (BstYI)/FastDigest® PsuI* (R↓GATCT)
Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDi-gest® DpnI, MboI/FastDigest® MboI, PsuI (BstYI)/FastDigest® PsuI
PsuI (BstYI)/FastDigest® PsuI* (R↓GATCC)BamHI/FastDigest® BamHI, Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspLI (NlaIV)/FastDigest® NlaIV (BspLI), BspPI (AlwI), DpnI/Fast-Digest® DpnI, MboI/FastDigest® MboI, PsuI (BstYI)/FastDigest® PsuI
BbeI (GGCGC↓C)FastDigest® HaeII (BfoI)* (RGCGC↓C)
BbeI, FastDigest® HaeII (BfoI), BshNI (BanI)/FastDigest® BanI (BshNI), BspLI (NlaIV)/FastDigest® NlaIV (BspLI), EheI (SfoI)/FastDigest® EheI, HhaI/FastDigest® HhaI, Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I), Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), NarI, SspDI (KasI)
FastDigest® HaeII (BfoI)* (RGCGC↓T)FastDigest® HaeII (BfoI), HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
Single letter codeR = G or A; H = A, C or T; Y = C or T; V = A, C or G;W = A or T; B = C, G or T;M = A or C; D = A, G or T;K = G or T; N = G, A, T or C.S = C or G;
Note* Restriction enzymes that have degenerate recognition
sequences (i.e., recognize more than one sequence). Be aware that these restriction endonucleases will cleave sequences in addition to the one listed.
• Fermentas FastDigest® enzymes are shown in ruby.• Fermentas conventional enzymes are shown in orange.
193
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzyme
Restriction enzyme that cleave the newly generated recognition sequence
BclI/FastDigest® BclI (T↓GATCA)
BamHI/FastDigest® BamHI (G↓GATCC), BglII/FastDigest® BglII (A↓GATCT), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I) (↓GATC), MboI/FastDigest® MboI (↓GATC), PsuI (BstYI)/FastDigest® PsuI* (R↓GATCC), PsuI (BstYI)/Fast-Digest® PsuI* (R↓GATCT)
Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
BcuI (SpeI)/FastDigest® SpeI (BcuI) (A↓CTAGT)
Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CTAGG), NheI/FastDigest® NheI (G↓CTAGC), XbaI/FastDigest® XbaI (T↓CTAGA), XmaJI (AvrII)/FastDi-gest® AvrII (XmaJI) (C↓CTAGG)
FspBI (BfaI)/FastDigest® BfaI (FspBI)
BfmI (SfcI)/FastDigest® SfcI (BfmI)* (C↓TGCAG)
Alw44I (ApaLI)/FastDigest® ApaLI (Alw44I) (G↓TGCAC) HpyCH4V
BglII/FastDigest® BglII (A↓GATCT)
BamHI/FastDigest® BamHI (G↓GATCC), PsuI (BstYI)/FastDigest® PsuI* (R↓GATCC)
Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PsuI (BstYI)/FastDigest® PsuI
BclI/FastDigest® BclI (T↓GATCA), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I) (↓GATC), MboI/FastDigest® MboI (↓GATC)
Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
PsuI (BstYI)/FastDigest® PsuI* (R↓GATCT)BglII/FastDigest® BglII, Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PsuI (BstYI)/FastDigest® PsuI
BmeT110I* (CC↓CGRG)
Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGCC), Hin6I (HinP1I)/FastDi-gest® HinP1I (Hin6I) (G↓CGC), NarI (GG↓CGCC), SsiI (AciI)/FastDigest® AciI (SsiI)* (C↓CGC)
SmuI (FauI), SsiI (AciI)/FastDigest® AciI (SsiI)
HpaII/FastDigest® HpaII (C↓CGG), MspI (HpaII)/FastDigest® MspI (C↓CGG), SsiI (AciI)/FastDigest® AciI (SsiI)* (G↓CGG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
BmeT110I* (CT↓CGRG)Bsp119I (BstBI)/FastDigest® Bsp119I (TT↓CGAA), Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I) (AT↓CGAT), TaqI/FastDigest® TaqI (T↓CGA), XmiI (AccI)/FastDi-gest® AccI (XmiI)* (GT↓CGAC)
TaqI/FastDigest® TaqI
BsaWI* (A↓CCGGW)
BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT), SgrAI* (CR↓CCGGTG)
BsaWI, BshTI (AgeI)/FastDigest® AgeI (BshTI), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), MreI (Sse232I)/Fast-Digest® MreI (CG↓CCGGCG), NgoMIV (G↓CCGGC), SgrAI* (CR↓CCGGCG)
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr9I (XmaI) (C↓CCGGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓CCGGG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA) BsaWI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
BsaWI* (T↓CCGGW)
BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT), SgrAI* (CR↓CCGGTG)
BsaWI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), MreI (Sse232I)/Fast-Digest® MreI (CG↓CCGGCG), NgoMIV (G↓CCGGC), SgrAI* (CR↓CCGGCG)
HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr9I (XmaI) (C↓CCGGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓CCGGG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA)BsaWI, HpaII/FastDigest® HpaII, Hpy188III, Kpn2I (BspEI)/FastDigest® Kpn2I, MspI (HpaII)/FastDigest® MspI
BseSI (Bme1580I)/FastDi-gest® Bme1580I (BseSI)* (GGGCA↓C)
SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GGGCA↓C)BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), SduI (Bsp1286I)/FastDi-gest® Bsp1286I (SduI)
BseSI (Bme1580I)/FastDi-gest® Bme1580I (BseSI)* (GGGCC↓C)
ApaI/FastDigest® ApaI (GGGCC↓C), Eco24I (BanII)* (GGGCC↓C), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GGGCC↓C)
ApaI/FastDigest® ApaI, BmgT120I, BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), Bsp120I (PspOMI)/FastDigest® Bsp120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
BseSI (Bme1580I)/FastDi-gest® Bme1580I (BseSI)* (GTGCA↓C)
Alw21I (BsiHKAI)/FastDigest® Alw21I* (GTGCA↓C), SduI (Bsp1286I)/FastDi-gest® Bsp1286I (SduI)* (GTGCA↓C)
Alw21I (BsiHKAI)/FastDigest® Alw21I, Alw44I (ApaLI)/FastDigest® ApaLI (Alw44I), BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), Hpy8I (MjaIV)/FastDigest® Hpy8I, HpyCH4V, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I) (ATGCA↓T) HpyCH4VPstI/FastDigest® PstI (CTGCA↓G), SdaI (SbfI)/FastDigest® SbfI (SdaI) (CCTGCA↓GG)
BsgI, HpyCH4V
BseSI (Bme1580I)/FastDi-gest® Bme1580I (BseSI)* (GTGCC↓C)
SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GTGCC↓C)BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), SduI (Bsp1286I)/FastDi-gest® Bsp1286I (SduI)
Bsh1285I (BsiEI)/FastDi-gest® BsiEI (Bsh1285I)* (CGAT↓CG)
PacI/FastDigest® PacI (TTAAT↓TAA) FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
PvuI/FastDigest® PvuI (CGAT↓CG), SfaAI (AsiSI)/FastDigest® AsiSI (SfaAI) (GCGAT↓CGC)
Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDi-gest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PvuI/FastDigest® PvuI
BshNI (BanI)/FastDigest® BanI (BshNI)* (G↓GCGCC)
SspDI (KasI) (G↓GCGCC)
BbeI, FastDigest® HaeII (BfoI), BshNI (BanI)/FastDigest® BanI (BshNI), BspLI (NlaIV)/FastDigest® NlaIV (BspLI), EheI (SfoI)/FastDigest® EheI, HhaI/FastDigest® HhaI, Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I), Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), NarI, SspDI (KasI)
BshNI (BanI)/FastDigest® BanI (BshNI)* (G↓GTACC)
Acc65I (Asp718I)/FastDigest® Acc65I (G↓GTACC)Acc65I (Asp718I)/FastDigest® Acc65I, BshNI (BanI)/FastDigest® BanI (BshNI), BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Csp6I (CviQI)/FastDigest® Csp6I, KpnI/FastDigest® KpnI, RsaI/FastDigest® RsaI
Bsp1407I (BsrGI)/FastDigest® Bsp1407I (T↓GTACA), Pfl23II (BsiWI)/FastDi-gest® BsiWI (Pfl23II) (C↓GTACG), TatI/FastDigest® TatI* (W↓GTACA), TatI/FastDigest® TatI* (W↓GTACT)
Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI
194
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzyme
Restriction enzyme that cleave the newly generated recognition sequence
BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT)
BsaWI* (W↓CCGGA), Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA) BsaWI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIBsaWI* (W↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT), SgrAI* (CR↓CCGGTG)
BsaWI, BshTI (AgeI)/FastDigest® AgeI (BshTI), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), MreI (Sse232I)/Fast-Digest® MreI (CG↓CCGGCG), NgoMIV (G↓CCGGC), SgrAI* (CR↓CCGGCG)
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr9I (XmaI) (C↓CCGGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓CCGGG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Bsp119I (BstBI)/FastDigest® Bsp119I (TT↓CGAA)
BmeT110I* (CY↓CGAG), Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I) (AT↓CGAT), TaqI/FastDigest® TaqI (T↓CGA), XmiI (AccI)/FastDigest® AccI (XmiI)* (GT↓CGAC)
TaqI/FastDigest® TaqI
Bsp120I (PspOMI)/FastDi-gest® Bsp120I (G↓GGCCC)
CfrI (EaeI)* (Y↓GGCCA), CfrI (EaeI)* (Y↓GGCCG), Eco52I (EagI)/FastDigest® EagI (Eco52I) (C↓GGCCG)
BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), CviJI
NotI/FastDigest® NotI (GC↓GGCCGC)BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/Fast-Digest® Sau96I (Cfr13I), CviJI, SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), SsiI (AciI)/FastDigest® AciI (SsiI), TauI/FastDigest® TauI
Bsp1407I (BsrGI)/FastDi-gest® Bsp1407I (T↓GTACA)
Acc65I (Asp718I)/FastDigest® Acc65I (G↓GTACC), BshNI (BanI)/FastDi-gest® BanI (BshNI)* (G↓GTACC), Pfl23II (BsiWI)/FastDigest® BsiWI (Pfl23II) (C↓GTACG)
Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI
TatI/FastDigest® TatI* (W↓GTACA)Bsp1407I (BsrGI)/FastDigest® Bsp1407I, Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI, TatI/FastDigest® TatI
TatI/FastDigest® TatI* (W↓GTACT)Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI, TatI/FastDigest® TatI
Bsp143I (Sau3AI)/FastDi-gest® Sau3AI (Bsp143I) (↓GATC)
BamHI/FastDigest® BamHI (G↓GATCC), BclI/FastDigest® BclI (T↓GATCA), BglII/FastDigest® BglII (A↓GATCT), MboI/FastDigest® MboI (↓GATC), PsuI (BstYI)/FastDigest® PsuI* (R↓GATCC), PsuI (BstYI)/FastDigest® PsuI* (R↓GATCT)
Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
BspTI (AflII)/FastDigest® AflII (BspTI) (C↓TTAAG)
SmoI (SmlI)* (C↓TTAAG)BspTI (AflII)/FastDigest® AflII (BspTI), FastDigest® MseI (SaqAI), SmoI (SmlI), Tru1I (MseI)/FastDigest® Tru1I
Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I) (AT↓CGAT)
BmeT110I* (CY↓CGAG), Bsp119I (BstBI)/FastDigest® Bsp119I (TT↓CGAA), TaqI/FastDigest® TaqI (T↓CGA), XmiI (AccI)/FastDigest® AccI (XmiI)* (GT↓CGAC)
TaqI/FastDigest® TaqI
BtgI* (C↓CACGG) AflIII* (A↓CACGT) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI
BtgI* (C↓CATGG)
AflIII* (A↓CATGT), FatI (↓CATG), PagI (BspHI)/FastDigest® BspHI (PagI) (T↓CATGA), PscI (PciI) (A↓CATGT)
CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)
Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CATGG), NcoI/FastDigest® NcoI (C↓CATGG)
BseDI (BsaJI)/FastDigest® BsaJI (BseDI), BtgI, CviAII, Eco130I (StyI)/FastDi-gest® StyI (Eco130I), FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), NcoI/FastDigest® NcoI
BtgI* (C↓CGCGG)AflIII* (A↓CGCGT), MluI/FastDigest® MluI (A↓CGCGT) Bsh1236I (BstUI)/FastDigest® Bsh1236I, SsiI (AciI)/FastDigest® AciI (SsiI)MauBI/FastDigest® MauBI (CG↓CGCGCG), SgsI (AscI)/FastDigest® AscI (SgsI) (GG↓CGCGCC), PauI (BssHII)/FastDigest® BssHII (PteI) (G↓CGCGC)
Bsh1236I (BstUI)/FastDigest® Bsh1236I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), SsiI (AciI)/FastDigest® AciI (SsiI)
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (A↓CCGGY)
BsaWI* (W↓CCGGA), Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA) BsaWI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIBsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), SgrAI* (CR↓CCGGTG)
BsaWI, BshTI (AgeI)/FastDigest® AgeI (BshTI), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr9I (XmaI) (C↓CCGGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓CCGGG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
MreI (Sse232I)/FastDigest® MreI (CG↓CCGGCG), NgoMIV (G↓CCGGC), SgrAI* (CR↓CCGGCG)
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (G↓CCGGY)
BsaWI* (W↓CCGGA), Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIBsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), SgrAI* (CR↓CCGGTG)
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr9I (XmaI) (C↓CCGGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓CCGGG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
MreI (Sse232I)/FastDigest® MreI (CG↓CCGGCG), NgoMIV (G↓CCGGC), SgrAI* (CR↓CCGGCG)
Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI)
Cfr42I (SacII) (CCGC↓GG) Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)* (CGGC↓CG) SsiI (AciI)/FastDigest® AciI (SsiI)
Cfr9I (XmaI) (C↓CCGGG)
BsaWI* (W↓CCGGA), BsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT), Kpn2I (BspEI)/FastDi-gest® Kpn2I (T↓CCGGA), MreI (Sse232I)/FastDigest® MreI (CG↓CCGGCG), NgoMIV (G↓CCGGC), SgrAI* (CR↓CCGGCG), SgrAI* (CR↓CCGGTG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓CCGGG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BmeT110I, BseDI (BsaJI)/FastDigest® BsaJI (BseDI), BssKI, Cfr9I (XmaI), Eco88I (AvaI)/FastDigest® AvaI (Eco88I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, SmaI/FastDigest® SmaI
195
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzyme
Restriction enzyme that cleave the newly generated recognition sequence
CfrI (EaeI)* (C↓GGCCR)
Bsp120I (PspOMI)/FastDigest® Bsp120I (G↓GGCCC)BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), CviJI
Eco52I (EagI)/FastDigest® EagI (Eco52I) (C↓GGCCG)Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I)
NotI/FastDigest® NotI (GC↓GGCCGC)
Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), SsiI (AciI)/FastDigest® AciI (SsiI), TauI/FastDigest® TauI
CfrI (EaeI)* (T↓GGCCR)
Bsp120I (PspOMI)/FastDigest® Bsp120I (G↓GGCCC)BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), CviJI
Eco52I (EagI)/FastDigest® EagI (Eco52I) (C↓GGCCG) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI
NotI/FastDigest® NotI (GC↓GGCCGC)BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), SsiI (AciI)/FastDigest® AciI (SsiI), TauI/FastDi-gest® TauI
CpoI (RsrII)/FastDigest® RsrII (CpoI)* (CG↓GACCG)
Eco47I (AvaII)/FastDigest® AvaII (Eco47I)* (G↓GACC)BmgT120I, Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I)
FastDigest® SanDI (KflI)* (GG↓GACCC), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GACCC)
BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I)
Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GACCT)BmgT120I, Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), SetI
CpoI (RsrII)/FastDigest® RsrII (CpoI)* (CG↓GTCCG)
Eco47I (AvaII)/FastDigest® AvaII (Eco47I)* (G↓GTCC), Psp5II (PpuMI)/FastDi-gest® PpuMI (Psp5II)* (RG↓GTCCT)
BmgT120I, Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I)
FastDigest® SanDI (KflI)* (GG↓GTCCC), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GTCCC)
BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I)
Csp6I (CviQI)/FastDigest® Csp6I (G↓TAC)
NdeI/FastDigest® NdeI (CA↓TATG) FaiI
CviAII (C↓ATG) XmiI (AccI)/FastDigest® AccI (XmiI)* (GT↓ATAC) FaiI
Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CATGG)
AflIII* (A↓CATGT), FatI (↓CATG), PagI (BspHI)/FastDigest® BspHI (PagI) (T↓CATGA), PscI (PciI) (A↓CATGT)
CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)
BtgI* (C↓CATGG), NcoI/FastDigest® NcoI (C↓CATGG)BseDI (BsaJI)/FastDigest® BsaJI (BseDI), BtgI, CviAII, Eco130I (StyI)/FastDi-gest® StyI (Eco130I), FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), NcoI/FastDigest® NcoI
Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CTAGG)
BcuI (SpeI)/FastDigest® SpeI (BcuI) (A↓CTAGT), NheI/FastDigest® NheI (G↓CTAGC), XbaI/FastDigest® XbaI (T↓CTAGA)
FspBI (BfaI)/FastDigest® BfaI (FspBI)
XmaJI (AvrII)/FastDigest® AvrII (XmaJI) (C↓CTAGG)BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Eco130I (StyI)/FastDigest® StyI (Eco130I), FspBI (BfaI)/FastDigest® BfaI (FspBI), XmaJI (AvrII)/FastDigest® AvrII (XmaJI)
Eco24I (BanII)* (GAGCC↓C) SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GAGCC↓C) CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
Eco24I (BanII)* (GAGCT↓C)Alw21I (BsiHKAI)/FastDigest® Alw21I* (GAGCT↓C), SacI/FastDigest® SacI (GAGCT↓C), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GAGCT↓C)
AluI/FastDigest® AluI, Alw21I (BsiHKAI)/FastDigest® Alw21I, CviJI, Ecl136II (EcoICRI)/FastDigest® Ecl136II, Eco24I (BanII), SacI/FastDigest® SacI, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI), SetI
SetI* (AGCT) AluI/FastDigest® AluI, CviJI, SetI
Eco24I (BanII)* (GGGCC↓C)ApaI/FastDigest® ApaI (GGGCC↓C), BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI)* (GGGCC↓C), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GGGCC↓C)
ApaI/FastDigest® ApaI, BmgT120I, BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), Bsp120I (PspOMI)/FastDigest® Bsp120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
Eco24I (BanII)* (GGGCT↓C) SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GGGCT↓C) CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
Eco47I (AvaII)/FastDigest® AvaII (Eco47I)* (G↓GACC)
CpoI (RsrII)/FastDigest® RsrII (CpoI)* (CG↓GACCG)BmgT120I, Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I)
FastDigest® SanDI (KflI)* (GG↓GACCC), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GACCC)
BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I)
Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GACCT)BmgT120I, Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), SetI
Eco47I (AvaII)/FastDigest® AvaII (Eco47I)* (G↓GTCC)
CpoI (RsrII)/FastDigest® RsrII (CpoI)* (CG↓GTCCG), Psp5II (PpuMI)/FastDi-gest® PpuMI (Psp5II)* (RG↓GTCCT)
BmgT120I, Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I)
FastDigest® SanDI (KflI)* (GG↓GTCCC), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GTCCC)
BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I)
Eco52I (EagI)/FastDigest® EagI (Eco52I) (C↓GGCCG)
Bsp120I (PspOMI)/FastDigest® Bsp120I (G↓GGCCC)BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), CviJI
CfrI (EaeI)* (Y↓GGCCA) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI
CfrI (EaeI)* (Y↓GGCCG)Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I)
NotI/FastDigest® NotI (GC↓GGCCGC)
Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), SsiI (AciI)/FastDigest® AciI (SsiI), TauI/FastDigest® TauI
196
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzyme
Restriction enzyme that cleave the newly generated recognition sequence
Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓CCGGG)
BsaWI* (W↓CCGGA), BsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT), Kpn2I (BspEI)/FastDi-gest® Kpn2I (T↓CCGGA), MreI (Sse232I)/FastDigest® MreI (CG↓CCGGCG), NgoMIV (G↓CCGGC), SgrAI* (CR↓CCGGCG), SgrAI* (CR↓CCGGTG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr9I (XmaI) (C↓CCGGG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BmeT110I, BseDI (BsaJI)/FastDigest® BsaJI (BseDI), BssKI, Cfr9I (XmaI), Eco88I (AvaI)/FastDigest® AvaI (Eco88I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, SmaI/FastDigest® SmaI
Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓TCGAG)
AbsI (CC↓TCGAGG), PspXI* (VC↓TCGAGC), PspXI* (VC↓TCGAGG), PspXI* (VC↓TCGAGT), SmoI (SmlI)* (C↓TCGAG), XhoI/FastDigest® XhoI (C↓TCGAG)
BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI
SalI/FastDigest® SalI (G↓TCGAC) TaqI/FastDigest® TaqISgrDI (CG↓TCGACG) Hpy99I, TaqI/FastDigest® TaqI
EcoRI/FastDigest® EcoRI (G↓AATTC)
MunI (MfeI)/FastDigest® MfeI (MunI) (C↓AATTG), TasI (Tsp509I)/FastDigest® Tsp509I (TasI) (↓AATT)
TasI (Tsp509I)/FastDigest® Tsp509I (TasI)
XapI (ApoI)/FastDigest® XapI* (R↓AATTC)EcoRI/FastDigest® EcoRI, TasI (Tsp509I)/FastDigest® Tsp509I (TasI), XapI (ApoI)/FastDigest® XapI
XapI (ApoI)/FastDigest® XapI* (R↓AATTT) TasI (Tsp509I)/FastDigest® Tsp509I (TasI), XapI (ApoI)/FastDigest® XapI
EcoRII* (↓CCAGG) FastDigest® SexAI (CsiI)* (A↓CCAGGT)Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, EcoRII, MvaI (BstNI)/FastDigest® MvaI, SetI
EcoRII* (↓CCTGG) FastDigest® SexAI (CsiI)* (A↓CCTGGT)Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, EcoRII, MvaI (BstNI)/FastDigest® MvaI
FatI (↓CATG)AflIII* (A↓CATGT), BtgI* (C↓CATGG), Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CATGG), NcoI/FastDigest® NcoI (C↓CATGG), PagI (BspHI)/FastDigest® BspHI (PagI) (T↓CATGA), PscI (PciI) (A↓CATGT)
CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)
FspBI (BfaI)/FastDigest® BfaI (FspBI) (C↓TAG)
NdeI/FastDigest® NdeI (CA↓TATG) FaiI
FastDigest® HaeII (BfoI)* (AGCGC↓Y)
BbeI (GGCGC↓C)FastDigest® HaeII (BfoI), HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
FastDigest® HaeII (BfoI)* (GGCGC↓Y)
BbeI (GGCGC↓C)
BbeI, FastDigest® HaeII (BfoI), BshNI (BanI)/FastDigest® BanI (BshNI), BspLI (NlaIV)/FastDigest® NlaIV (BspLI), EheI (SfoI)/FastDigest® EheI, HhaI/FastDigest® HhaI, Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I), Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), NarI, SspDI (KasI)
Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GA↓CGYC)
Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I) (G↓CGC), SsiI (AciI)/FastDigest® AciI (SsiI)* (C↓CGC)
CseI (HgaI)/FastDigest® HgaI (CseI)
MaeII (A↓CGT), Psp1406I (AclI)/FastDigest® AclI (Psp1406I) (AA↓CGTT) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiINarI (GG↓CGCC) CseI (HgaI)/FastDigest® HgaI (CseI), Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)
Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GG↓CGYC)
Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I) (G↓CGC), SsiI (AciI)/FastDigest® AciI (SsiI)* (C↓CGC)
HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
NarI (GG↓CGCC)
BbeI, FastDigest® HaeII (BfoI), BshNI (BanI)/FastDigest® BanI (BshNI), BspLI (NlaIV)/FastDigest® NlaIV (BspLI), EheI (SfoI)/FastDigest® EheI, HhaI/FastDigest® HhaI, Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I), Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), NarI, SspDI (KasI)
Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II) (CATG↓)
PaeI (SphI)/FastDigest® SphI (PaeI) (GCATG↓C), XceI (NspI)/FastDigest® NspI (XceI)* (RCATG↓C), XceI (NspI)/FastDigest® NspI (XceI)* (RCATG↓T)
CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)
Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I) (G↓CGC)
Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGCC), NarI (GG↓CGCC), SsiI (AciI)/FastDigest® AciI (SsiI)* (C↓CGC)
HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
HpaII/FastDigest® HpaII (C↓CGG)
BmeT110I* (CY↓CGGG)BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGCC), Hin6I (HinP1I)/FastDi-gest® HinP1I (Hin6I) (G↓CGC), NarI (GG↓CGCC), SsiI (AciI)/FastDigest® AciI (SsiI)* (C↓CGC)
SsiI (AciI)/FastDigest® AciI (SsiI)
MspI (HpaII)/FastDigest® MspI (C↓CGG), SsiI (AciI)/FastDigest® AciI (SsiI)* (G↓CGG)
HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA)
BsaWI* (W↓CCGGA)BsaWI, HpaII/FastDigest® HpaII, Hpy188III, Kpn2I (BspEI)/FastDigest® Kpn2I, MspI (HpaII)/FastDigest® MspI
BsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT), SgrAI* (CR↓CCGGTG)
BsaWI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), MreI (Sse232I)/Fast-Digest® MreI (CG↓CCGGCG), NgoMIV (G↓CCGGC), SgrAI* (CR↓CCGGCG)
HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr9I (XmaI) (C↓CCGGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓CCGGG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
MaeII (A↓CGT)Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGTC), Psp1406I (AclI)/FastDi-gest® AclI (Psp1406I) (AA↓CGTT)
MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI
MauBI/FastDigest® MauBI (CG↓CGCGCG)
AflIII* (A↓CGCGT), BtgI* (C↓CGCGG), MluI/FastDigest® MluI (A↓CGCGT)Bsh1236I (BstUI)/FastDigest® Bsh1236I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
PauI (BssHII)/FastDigest® BssHII (PteI) (G↓CGCGC), SgsI (AscI)/FastDigest® AscI (SgsI) (GG↓CGCGCC)
Bsh1236I (BstUI)/FastDigest® Bsh1236I, Cac8I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), PauI (BssHII)/FastDigest® BssHII (PteI)
197
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzyme
Restriction enzyme that cleave the newly generated recognition sequence
MboI/FastDigest® MboI (↓GATC)
BamHI/FastDigest® BamHI (G↓GATCC), BclI/FastDigest® BclI (T↓GATCA), BglII/FastDigest® BglII (A↓GATCT), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I) (↓GATC), PsuI (BstYI)/FastDigest® PsuI* (R↓GATCC), PsuI (BstYI)/FastDigest® PsuI* (R↓GATCT)
Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
MluI/FastDigest® MluI (A↓CGCGT)
AflIII* (A↓CGCGT) AflIII, Bsh1236I (BstUI)/FastDigest® Bsh1236I, MluI/FastDigest® MluIBtgI* (C↓CGCGG) Bsh1236I (BstUI)/FastDigest® Bsh1236IMauBI/FastDigest® MauBI (CG↓CGCGCG), SgsI (AscI)/FastDigest® AscI (SgsI) (GG↓CGCGCC), PauI (BssHII)/FastDigest® BssHII (PteI) (G↓CGCGC)
Bsh1236I (BstUI)/FastDigest® Bsh1236I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I) (ATGCA↓T)
Alw21I (BsiHKAI)/FastDigest® Alw21I* (GTGCA↓C), BseSI (Bme1580I)/Fast-Digest® Bme1580I (BseSI)* (GTGCA↓C), PstI/FastDigest® PstI (CTGCA↓G), SdaI (SbfI)/FastDigest® SbfI (SdaI) (CCTGCA↓GG), SduI (Bsp1286I)/FastDi-gest® Bsp1286I (SduI)* (GTGCA↓C)
HpyCH4V
MreI (Sse232I)/FastDigest® MreI (CG↓CCGGCG)
BsaWI* (W↓CCGGA), Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIBsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT)
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), NgoMIV (G↓CCGGC)Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI)
Cfr9I (XmaI) (C↓CCGGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓CCGGG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
SgrAI* (CR↓CCGGCG)Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MreI (Sse232I)/FastDigest® MreI, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI), SgrAI
SgrAI* (CR↓CCGGTG)Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, SgrAI
FastDigest® MseI (SaqAI) (T↓TAA)
NdeI/FastDigest® NdeI (CA↓TATG) FaiITru1I (MseI)/FastDigest® Tru1I (T↓TAA), VspI (AseI)/FastDigest® AseI (VspI) (AT↓TAAT)
FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
MspI (HpaII)/FastDigest® MspI (C↓CGG)
BmeT110I* (CY↓CGGG)BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGCC), Hin6I (HinP1I)/FastDi-gest® HinP1I (Hin6I) (G↓CGC), NarI (GG↓CGCC), SsiI (AciI)/FastDigest® AciI (SsiI)* (C↓CGC)
SsiI (AciI)/FastDigest® AciI (SsiI)
HpaII/FastDigest® HpaII (C↓CGG), SsiI (AciI)/FastDigest® AciI (SsiI)* (G↓CGG)
HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
MunI (MfeI)/FastDigest® MfeI (MunI) (C↓AATTG)
EcoRI/FastDigest® EcoRI (G↓AATTC), TasI (Tsp509I)/FastDigest® Tsp509I (TasI) (↓AATT), XapI (ApoI)/FastDigest® XapI* (R↓AATTC), XapI (ApoI)/FastDi-gest® XapI* (R↓AATTT)
TasI (Tsp509I)/FastDigest® Tsp509I (TasI)
NarI (GG↓CGCC)
Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGCC)
BbeI, FastDigest® HaeII (BfoI), BshNI (BanI)/FastDigest® BanI (BshNI), BspLI (NlaIV)/FastDigest® NlaIV (BspLI), EheI (SfoI)/FastDigest® EheI, HhaI/FastDigest® HhaI, Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I), Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), NarI, SspDI (KasI)
Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGTC) Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I) (G↓CGC), SsiI (AciI)/FastDigest® AciI (SsiI)* (C↓CGC)
HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
NcoI/FastDigest® NcoI (C↓CATGG)
AflIII* (A↓CATGT), FatI (↓CATG), PagI (BspHI)/FastDigest® BspHI (PagI) (T↓CATGA), PscI (PciI) (A↓CATGT)
CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)
BtgI* (C↓CATGG), Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CATGG)BseDI (BsaJI)/FastDigest® BsaJI (BseDI), BtgI, CviAII, Eco130I (StyI)/FastDi-gest® StyI (Eco130I), FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), NcoI/FastDigest® NcoI
NdeI/FastDigest® NdeI (CA↓TATG)
Csp6I (CviQI)/FastDigest® Csp6I (G↓TAC), FspBI (BfaI)/FastDigest® BfaI (FspBI) (C↓TAG), FastDigest® MseI (SaqAI) (T↓TAA), Tru1I (MseI)/FastDigest® Tru1I (T↓TAA), VspI (AseI)/FastDigest® AseI (VspI) (AT↓TAAT)
FaiI
NgoMIV (G↓CCGGC)
BsaWI* (W↓CCGGA), Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIBsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT), SgrAI* (CR↓CCGGTG)
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), MreI (Sse232I)/Fast-Digest® MreI (CG↓CCGGCG), SgrAI* (CR↓CCGGCG)
Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI)
Cfr9I (XmaI) (C↓CCGGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓CCGGG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
NheI/FastDigest® NheI (G↓CTAGC)
BcuI (SpeI)/FastDigest® SpeI (BcuI) (A↓CTAGT), Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CTAGG), XbaI/FastDigest® XbaI (T↓CTAGA), XmaJI (AvrII)/FastDigest® AvrII (XmaJI) (C↓CTAGG)
FspBI (BfaI)/FastDigest® BfaI (FspBI)
NotI/FastDigest® NotI (GC↓GGCCGC)
Bsp120I (PspOMI)/FastDigest® Bsp120I (G↓GGCCC)BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), CviJI, SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), TauI/FastDigest® TauI
CfrI (EaeI)* (Y↓GGCCA)BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), TauI/FastDigest® TauI
CfrI (EaeI)* (Y↓GGCCG), Eco52I (EagI)/FastDigest® EagI (Eco52I) (C↓GGCCG)Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), TauI/FastDigest® TauI
198
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzyme
Restriction enzyme that cleave the newly generated recognition sequence
PacI/FastDigest® PacI (TTAAT↓TAA)
Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)* (CGAT↓CG), PvuI/FastDi-gest® PvuI (CGAT↓CG), SfaAI (AsiSI)/FastDigest® AsiSI (SfaAI) (GCGAT↓CGC)
FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
PaeI (SphI)/FastDigest® SphI (PaeI) (GCATG↓C)
Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II) (CATG) CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)
XceI (NspI)/FastDigest® NspI (XceI)* (RCATG↓C)Cac8I, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), PaeI (SphI)/FastDigest® SphI (PaeI), XceI (NspI)/FastDigest® NspI (XceI)
XceI (NspI)/FastDigest® NspI (XceI)* (RCATG↓T)CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), XceI (NspI)/FastDi-gest® NspI (XceI)
PagI (BspHI)/FastDigest® BspHI (PagI) (T↓CATGA)
AflIII* (A↓CATGT), BtgI* (C↓CATGG), Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CATGG), FatI (↓CATG), NcoI/FastDigest® NcoI (C↓CATGG), PscI (PciI) (A↓CATGT)
CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)
PasI* (CC↓CAGGG) TseI* (G↓CAGC) BseYI
PauI (BssHII)/FastDigest® BssHII (PteI) (G↓CGCGC)
AflIII* (A↓CGCGT), BtgI* (C↓CGCGG), MluI/FastDigest® MluI (A↓CGCGT)Bsh1236I (BstUI)/FastDigest® Bsh1236I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
MauBI/FastDigest® MauBI (CG↓CGCGCG), SgsI (AscI)/FastDigest® AscI (SgsI) (GG↓CGCGCC)
Bsh1236I (BstUI)/FastDigest® Bsh1236I, Cac8I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), PauI (BssHII)/FastDigest® BssHII (PteI)
Pfl23II (BsiWI)/FastDigest® BsiWI (Pfl23II) (C↓GTACG)
Acc65I (Asp718I)/FastDigest® Acc65I (G↓GTACC), BshNI (BanI)/FastDigest® BanI (BshNI)* (G↓GTACC), Bsp1407I (BsrGI)/FastDigest® Bsp1407I (T↓GTACA), TatI/FastDigest® TatI* (W↓GTACA), TatI/FastDigest® TatI* (W↓GTACT)
Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI
PscI (PciI) (A↓CATGT)
AflIII* (A↓CATGT)AflIII, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), PscI (PciI), XceI (NspI)/FastDigest® NspI (XceI)
BtgI* (C↓CATGG), Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CATGG), FatI (↓CATG), NcoI/FastDigest® NcoI (C↓CATGG), PagI (BspHI)/FastDigest® BspHI (PagI) (T↓CATGA)
CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)
Psp1406I (AclI)/FastDigest® AclI (Psp1406I) (AA↓CGTT)
Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGTC), MaeII (A↓CGT) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI
Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (AG↓GACCY)
CpoI (RsrII)/FastDigest® RsrII (CpoI)* (CG↓GACCG), Eco47I (AvaII)/FastDi-gest® AvaII (Eco47I)* (G↓GACC)
BmgT120I, Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I)
FastDigest® SanDI (KflI)* (GG↓GACCC)BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), EcoO109I (DraII)/FastDigest® EcoO109I, Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)
Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (AG↓GTCCY)
CpoI (RsrII)/FastDigest® RsrII (CpoI)* (CG↓GTCCG), Eco47I (AvaII)/FastDi-gest® AvaII (Eco47I)* (G↓GTCC)
BmgT120I, Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), SetI
FastDigest® SanDI (KflI)* (GG↓GTCCC)
BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), EcoO109I (DraII)/FastDigest® EcoO109I, Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II), SetI
Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (GG↓GACCY)
CpoI (RsrII)/FastDigest® RsrII (CpoI)* (CG↓GACCG), Eco47I (AvaII)/FastDi-gest® AvaII (Eco47I)* (G↓GACC)
BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/Fast-Digest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), FaqI (BsmFI)/FastDigest® BsmFI (FaqI)
FastDigest® SanDI (KflI)* (GG↓GACCC)
BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), EcoO109I (DraII)/FastDigest® EcoO109I, FaqI (BsmFI)/FastDigest® BsmFI (FaqI), FastDigest® SanDI (KflI), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)
Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (GG↓GTCCY)
CpoI (RsrII)/FastDigest® RsrII (CpoI)* (CG↓GTCCG), Eco47I (AvaII)/FastDi-gest® AvaII (Eco47I)* (G↓GTCC)
BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I)
FastDigest® SanDI (KflI)* (GG↓GTCCC)
BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), EcoO109I (DraII)/FastDigest® EcoO109I, FastDigest® SanDI (KflI), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)
PspXI* (AC↓TCGAGB)
AbsI (CC↓TCGAGG)BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), PspXI, SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI
Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓TCGAG), SmoI (SmlI)* (C↓TCGAG), XhoI/FastDigest® XhoI (C↓TCGAG)
BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI
SalI/FastDigest® SalI (G↓TCGAC) TaqI/FastDigest® TaqISgrDI (CG↓TCGACG) Hpy99I, TaqI/FastDigest® TaqI
PspXI* (CC↓TCGAGB)
AbsI (CC↓TCGAGG)AbsI, BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), MnlI/FastDigest® MnlI, PspXI, SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI
Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓TCGAG), SmoI (SmlI)* (C↓TCGAG), XhoI/FastDigest® XhoI (C↓TCGAG)
BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), MnlI/FastDigest® MnlI, SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI
SalI/FastDigest® SalI (G↓TCGAC) MnlI/FastDigest® MnlI, TaqI/FastDigest® TaqISgrDI (CG↓TCGACG) Hpy99I, MnlI/FastDigest® MnlI, TaqI/FastDigest® TaqI
PspXI* (GC↓TCGAGB)
AbsI (CC↓TCGAGG)BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), PspXI, SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI
Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓TCGAG), SmoI (SmlI)* (C↓TCGAG), XhoI/FastDigest® XhoI (C↓TCGAG)
BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI
SalI/FastDigest® SalI (G↓TCGAC) TaqI/FastDigest® TaqISgrDI (CG↓TCGACG) Hpy99I, TaqI/FastDigest® TaqI
199
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzyme
Restriction enzyme that cleave the newly generated recognition sequence
PstI/FastDigest® PstI (CTGCA↓G)
Alw21I (BsiHKAI)/FastDigest® Alw21I* (GTGCA↓C), BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI)* (GTGCA↓C), Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I) (ATGCA↓T), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GTGCA↓C)
HpyCH4V
SdaI (SbfI)/FastDigest® SbfI (SdaI) (CCTGCA↓GG) BfmI (SfcI)/FastDigest® SfcI (BfmI), HpyCH4V, PstI/FastDigest® PstI
PsuI (BstYI)/FastDigest® PsuI* (A↓GATCY)
BamHI/FastDigest® BamHI (G↓GATCC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PsuI (BstYI)/FastDigest® PsuI
BclI/FastDigest® BclI (T↓GATCA), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I) (↓GATC), MboI/FastDigest® MboI (↓GATC)
Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI
BglII/FastDigest® BglII (A↓GATCT)BglII/FastDigest® BglII, Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PsuI (BstYI)/FastDigest® PsuI
PsuI (BstYI)/FastDigest® PsuI* (G↓GATCY)
BamHI/FastDigest® BamHI (G↓GATCC)BamHI/FastDigest® BamHI, Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspLI (NlaIV)/FastDigest® NlaIV (BspLI), BspPI (AlwI), DpnI/Fast-Digest® DpnI, MboI/FastDigest® MboI, PsuI (BstYI)/FastDigest® PsuI
BclI/FastDigest® BclI (T↓GATCA), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I) (↓GATC), MboI/FastDigest® MboI (↓GATC)
Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDi-gest® DpnI, MboI/FastDigest® MboI
BglII/FastDigest® BglII (A↓GATCT)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDi-gest® DpnI, MboI/FastDigest® MboI, PsuI (BstYI)/FastDigest® PsuI
PvuI/FastDigest® PvuI (CGAT↓CG)
Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)* (CGAT↓CG), SfaAI (AsiSI)/FastDigest® AsiSI (SfaAI) (GCGAT↓CGC)
Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDi-gest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PvuI/FastDigest® PvuI
PacI/FastDigest® PacI (TTAAT↓TAA) FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
SacI/FastDigest® SacI (GAGCT↓C)
Alw21I (BsiHKAI)/FastDigest® Alw21I* (GAGCT↓C), Eco24I (BanII)* (GAGCT↓C), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GAGCT↓C)
AluI/FastDigest® AluI, Alw21I (BsiHKAI)/FastDigest® Alw21I, CviJI, Ecl136II (EcoICRI)/FastDigest® Ecl136II, Eco24I (BanII), SacI/FastDigest® SacI, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI), SetI
SetI* (AGCT) AluI/FastDigest® AluI, CviJI, SetI
SalI/FastDigest® SalI (G↓TCGAC)
AbsI (CC↓TCGAGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓TCGAG), PspXI* (VC↓TCGAGC), PspXI* (VC↓TCGAGG), PspXI* (VC↓TCGAGT), SmoI (SmlI)* (C↓TCGAG), XhoI/FastDigest® XhoI (C↓TCGAG)
TaqI/FastDigest® TaqI
SgrDI (CG↓TCGACG)HincII (HindII)/FastDigest® HincII, Hpy8I (MjaIV)/FastDigest® Hpy8I, Hpy99I, SalI/FastDigest® SalI, TaqI/FastDigest® TaqI, XmiI (AccI)/FastDigest® AccI (XmiI)
FastDigest® SanDI (KflI)* (GG↓GACCC)
CpoI (RsrII)/FastDigest® RsrII (CpoI)* (CG↓GACCG), Eco47I (AvaII)/FastDi-gest® AvaII (Eco47I)* (G↓GACC)
BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/Fast-Digest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), FaqI (BsmFI)/FastDigest® BsmFI (FaqI)
Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GACCC)
BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), EcoO109I (DraII)/FastDigest® EcoO109I, FaqI (BsmFI)/FastDigest® BsmFI (FaqI), FastDigest® SanDI (KflI), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)
Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GACCT)
BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), EcoO109I (DraII)/FastDigest® EcoO109I, FaqI (BsmFI)/FastDigest® BsmFI (FaqI), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II), SetI
FastDigest® SanDI (KflI)* (GG↓GTCCC)
CpoI (RsrII)/FastDigest® RsrII (CpoI)* (CG↓GTCCG), Eco47I (AvaII)/FastDi-gest® AvaII (Eco47I)* (G↓GTCC)
BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I)
Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GTCCC)
BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), EcoO109I (DraII)/FastDigest® EcoO109I, FastDigest® SanDI (KflI), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)
Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GTCCT)BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), EcoO109I (DraII)/FastDigest® EcoO109I, Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)
SdaI (SbfI)/FastDigest® SbfI (SdaI) (CCTGCA↓GG)
Alw21I (BsiHKAI)/FastDigest® Alw21I* (GTGCA↓C), BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI)* (GTGCA↓C), Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I) (ATGCA↓T), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GTGCA↓C)
HpyCH4V
PstI/FastDigest® PstI (CTGCA↓G) BfmI (SfcI)/FastDigest® SfcI (BfmI), HpyCH4V, PstI/FastDigest® PstISduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GAGCA↓C)
Alw21I (BsiHKAI)/FastDigest® Alw21I* (GAGCA↓C)Alw21I (BsiHKAI)/FastDigest® Alw21I, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GAGCC↓C)
Eco24I (BanII)* (GAGCC↓C) CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GAGCT↓C)
Alw21I (BsiHKAI)/FastDigest® Alw21I* (GAGCT↓C), Eco24I (BanII)* (GAGCT↓C), SacI/FastDigest® SacI (GAGCT↓C)
AluI/FastDigest® AluI, Alw21I (BsiHKAI)/FastDigest® Alw21I, CviJI, Ecl136II (EcoICRI)/FastDigest® Ecl136II, Eco24I (BanII), SacI/FastDigest® SacI, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI), SetI
SetI* (AGCT) AluI/FastDigest® AluI, CviJI, SetISduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GGGCA↓C)
BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI)* (GGGCA↓C)BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), SduI (Bsp1286I)/FastDi-gest® Bsp1286I (SduI)
200
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends.
(continued on next page)
First restriction enzyme
Second restriction enzyme
Restriction enzyme that cleave the newly generated recognition sequence
SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GGGCC↓C)
ApaI/FastDigest® ApaI (GGGCC↓C), BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI)* (GGGCC↓C), Eco24I (BanII)* (GGGCC↓C)
ApaI/FastDigest® ApaI, BmgT120I, BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), Bsp120I (PspOMI)/FastDigest® Bsp120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GGGCT↓C)
Eco24I (BanII)* (GGGCT↓C) CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GTGCA↓C)
Alw21I (BsiHKAI)/FastDigest® Alw21I* (GTGCA↓C), BseSI (Bme1580I)/Fast-Digest® Bme1580I (BseSI)* (GTGCA↓C)
Alw21I (BsiHKAI)/FastDigest® Alw21I, Alw44I (ApaLI)/FastDigest® ApaLI (Alw44I), BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), Hpy8I (MjaIV)/FastDigest® Hpy8I, HpyCH4V, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I) (ATGCA↓T) HpyCH4VPstI/FastDigest® PstI (CTGCA↓G), SdaI (SbfI)/FastDigest® SbfI (SdaI) (CCTGCA↓GG)
BsgI, HpyCH4V
SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GTGCC↓C)
BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI)* (GTGCC↓C)BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), SduI (Bsp1286I)/FastDi-gest® Bsp1286I (SduI)
SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GTGCT↓C)
Alw21I (BsiHKAI)/FastDigest® Alw21I* (GTGCT↓C)Alw21I (BsiHKAI)/FastDigest® Alw21I, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)
SetI* (ACGT) AatII/FastDigest® AatII (GACGT↓C), TaiI (MaeII)/FastDigest® TaiI (ACGT) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI
SetI* (AGCT)Alw21I (BsiHKAI)/FastDigest® Alw21I* (GAGCT↓C), Eco24I (BanII)* (GAGCT↓C), SacI/FastDigest® SacI (GAGCT↓C), SduI (Bsp1286I)/FastDi-gest® Bsp1286I (SduI)* (GAGCT↓C)
AluI/FastDigest® AluI, CviJI, SetI
FastDigest® SexAI (CsiI)* (A↓CCAGGT)
EcoRII* (↓CCAGG)Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, EcoRII, MvaI (BstNI)/FastDigest® MvaI
FastDigest® SexAI (CsiI)* (A↓CCTGGT)
EcoRII* (↓CCTGG)Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, EcoRII, MvaI (BstNI)/FastDigest® MvaI, SetI
SfaAI (AsiSI)/FastDigest® AsiSI (SfaAI) (GCGAT↓CGC)
Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)* (CGAT↓CG), PvuI/FastDi-gest® PvuI (CGAT↓CG)
Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDi-gest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PvuI/FastDigest® PvuI
PacI/FastDigest® PacI (TTAAT↓TAA) FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
SgrAI* (CA↓CCGGYG)
BsaWI* (W↓CCGGA), Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA) BsaWI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIBsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT)
BsaWI, BshTI (AgeI)/FastDigest® AgeI (BshTI), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), NgoMIV (G↓CCGGC)Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr9I (XmaI) (C↓CCGGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓CCGGG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
MreI (Sse232I)/FastDigest® MreI (CG↓CCGGCG)Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, SgrAI
SgrAI* (CG↓CCGGYG)
BsaWI* (W↓CCGGA), Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIBsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT)
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), NgoMIV (G↓CCGGC)Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI)
Cfr9I (XmaI) (C↓CCGGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓CCGGG)
BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
MreI (Sse232I)/FastDigest® MreI (CG↓CCGGCG)Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MreI (Sse232I)/FastDigest® MreI, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI), SgrAI
SgrDI (CG↓TCGACG)
AbsI (CC↓TCGAGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓TCGAG), PspXI* (VC↓TCGAGC), PspXI* (VC↓TCGAGG), PspXI* (VC↓TCGAGT), SmoI (SmlI)* (C↓TCGAG), XhoI/FastDigest® XhoI (C↓TCGAG)
Hpy99I, TaqI/FastDigest® TaqI
SalI/FastDigest® SalI (G↓TCGAC)HincII (HindII)/FastDigest® HincII, Hpy8I (MjaIV)/FastDigest® Hpy8I, Hpy99I, SalI/FastDigest® SalI, TaqI/FastDigest® TaqI, XmiI (AccI)/FastDigest® AccI (XmiI)
SgsI (AscI)/FastDigest® AscI (SgsI) (GG↓CGCGCC)
AflIII* (A↓CGCGT), BtgI* (C↓CGCGG), MluI/FastDigest® MluI (A↓CGCGT)Bsh1236I (BstUI)/FastDigest® Bsh1236I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
MauBI/FastDigest® MauBI (CG↓CGCGCG), PauI (BssHII)/FastDigest® BssHII (PteI) (G↓CGCGC)
Bsh1236I (BstUI)/FastDigest® Bsh1236I, Cac8I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), PauI (BssHII)/FastDigest® BssHII (PteI)
SmoI (SmlI)* (C↓TCGAG)
AbsI (CC↓TCGAGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓TCGAG), PspXI* (VC↓TCGAGC), PspXI* (VC↓TCGAGG), PspXI* (VC↓TCGAGT), XhoI/FastDigest® XhoI (C↓TCGAG)
BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI
SalI/FastDigest® SalI (G↓TCGAC) TaqI/FastDigest® TaqISgrDI (CG↓TCGACG) Hpy99I, TaqI/FastDigest® TaqI
SmoI (SmlI)* (C↓TTAAG) BspTI (AflII)/FastDigest® AflII (BspTI) (C↓TTAAG)BspTI (AflII)/FastDigest® AflII (BspTI), FastDigest® MseI (SaqAI), SmoI (SmlI), Tru1I (MseI)/FastDigest® Tru1I
SsiI (AciI)/FastDigest® AciI (SsiI)* (C↓CGC)
BmeT110I* (CY↓CGGG)BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGCC), Hin6I (HinP1I)/FastDi-gest® HinP1I (Hin6I) (G↓CGC), NarI (GG↓CGCC)
SsiI (AciI)/FastDigest® AciI (SsiI)
HpaII/FastDigest® HpaII (C↓CGG), MspI (HpaII)/FastDigest® MspI (C↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
201
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends.
First restriction enzyme
Second restriction enzyme
Restriction enzyme that cleave the newly generated recognition sequence
SsiI (AciI)/FastDigest® AciI (SsiI)* (G↓CGG)
Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGCC), Hin6I (HinP1I)/FastDi-gest® HinP1I (Hin6I) (G↓CGC), NarI (GG↓CGCC)
HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
SspDI (KasI) (G↓GCGCC) BshNI (BanI)/FastDigest® BanI (BshNI)* (G↓GCGCC)
BbeI, FastDigest® HaeII (BfoI), BshNI (BanI)/FastDigest® BanI (BshNI), BspLI (NlaIV)/FastDigest® NlaIV (BspLI), EheI (SfoI)/FastDigest® EheI, HhaI/FastDigest® HhaI, Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I), Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), NarI, SspDI (KasI)
TaiI (MaeII)/FastDigest® TaiI (ACGT)
AatII/FastDigest® AatII (GACGT↓C), SetI* (ACGT) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI
TaqI/FastDigest® TaqI (T↓CGA)
BmeT110I* (CY↓CGAG), Bsp119I (BstBI)/FastDigest® Bsp119I (TT↓CGAA), Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I) (AT↓CGAT), XmiI (AccI)/FastDigest® AccI (XmiI)* (GT↓CGAC)
TaqI/FastDigest® TaqI
TasI (Tsp509I)/FastDigest® Tsp509I (TasI) (↓AATT)
EcoRI/FastDigest® EcoRI (G↓AATTC), MunI (MfeI)/FastDigest® MfeI (MunI) (C↓AATTG), XapI (ApoI)/FastDigest® XapI* (R↓AATTC), XapI (ApoI)/FastDi-gest® XapI* (R↓AATTT)
TasI (Tsp509I)/FastDigest® Tsp509I (TasI)
TatI/FastDigest® TatI* (A↓GTACW)
Acc65I (Asp718I)/FastDigest® Acc65I (G↓GTACC), BshNI (BanI)/FastDi-gest® BanI (BshNI)* (G↓GTACC), Pfl23II (BsiWI)/FastDigest® BsiWI (Pfl23II) (C↓GTACG)
Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI
Bsp1407I (BsrGI)/FastDigest® Bsp1407I (T↓GTACA)Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI, TatI/FastDigest® TatI
TatI/FastDigest® TatI* (T↓GTACW)
Acc65I (Asp718I)/FastDigest® Acc65I (G↓GTACC), BshNI (BanI)/FastDi-gest® BanI (BshNI)* (G↓GTACC), Pfl23II (BsiWI)/FastDigest® BsiWI (Pfl23II) (C↓GTACG)
Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI
Bsp1407I (BsrGI)/FastDigest® Bsp1407I (T↓GTACA)Bsp1407I (BsrGI)/FastDigest® Bsp1407I, Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI, TatI/FastDigest® TatI
Tru1I (MseI)/FastDigest® Tru1I (T↓TAA)
NdeI/FastDigest® NdeI (CA↓TATG) FaiIFastDigest® MseI (SaqAI) (T↓TAA), VspI (AseI)/FastDigest® AseI (VspI) (AT↓TAAT)
FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
VspI (AseI)/FastDigest® AseI (VspI) (AT↓TAAT)
NdeI/FastDigest® NdeI (CA↓TATG) FaiIFastDigest® MseI (SaqAI) (T↓TAA), Tru1I (MseI)/FastDigest® Tru1I (T↓TAA) FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I
XapI (ApoI)/FastDigest® XapI* (A↓AATTY)
EcoRI/FastDigest® EcoRI (G↓AATTC) TasI (Tsp509I)/FastDigest® Tsp509I (TasI), XapI (ApoI)/FastDigest® XapIMunI (MfeI)/FastDigest® MfeI (MunI) (C↓AATTG), TasI (Tsp509I)/FastDigest® Tsp509I (TasI) (↓AATT)
TasI (Tsp509I)/FastDigest® Tsp509I (TasI)
XapI (ApoI)/FastDigest® XapI* (G↓AATTY)
EcoRI/FastDigest® EcoRI (G↓AATTC)EcoRI/FastDigest® EcoRI, TasI (Tsp509I)/FastDigest® Tsp509I (TasI), XapI (ApoI)/FastDigest® XapI
MunI (MfeI)/FastDigest® MfeI (MunI) (C↓AATTG), TasI (Tsp509I)/FastDigest® Tsp509I (TasI) (↓AATT)
TasI (Tsp509I)/FastDigest® Tsp509I (TasI)
XbaI/FastDigest® XbaI (T↓CTAGA)
BcuI (SpeI)/FastDigest® SpeI (BcuI) (A↓CTAGT), Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CTAGG), NheI/FastDigest® NheI (G↓CTAGC), XmaJI (AvrII)/FastDigest® AvrII (XmaJI) (C↓CTAGG)
FspBI (BfaI)/FastDigest® BfaI (FspBI)
XceI (NspI)/FastDigest® NspI (XceI)* (ACATG↓Y)
Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II) (CATG) CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)
PaeI (SphI)/FastDigest® SphI (PaeI) (GCATG↓C)CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), XceI (NspI)/FastDi-gest® NspI (XceI)
XceI (NspI)/FastDigest® NspI (XceI)* (GCATG↓Y)
Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II) (CATG) CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)
PaeI (SphI)/FastDigest® SphI (PaeI) (GCATG↓C)Cac8I, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), PaeI (SphI)/FastDigest® SphI (PaeI), XceI (NspI)/FastDigest® NspI (XceI)
XhoI/FastDigest® XhoI (C↓TCGAG)
AbsI (CC↓TCGAGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓TCGAG), PspXI* (VC↓TCGAGC), PspXI* (VC↓TCGAGG), PspXI* (VC↓TCGAGT), SmoI (SmlI)* (C↓TCGAG)
BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI
SalI/FastDigest® SalI (G↓TCGAC) TaqI/FastDigest® TaqISgrDI (CG↓TCGACG) Hpy99I, TaqI/FastDigest® TaqI
XmaJI (AvrII)/FastDigest® AvrII (XmaJI) (C↓CTAGG)
BcuI (SpeI)/FastDigest® SpeI (BcuI) (A↓CTAGT), NheI/FastDigest® NheI (G↓CTAGC), XbaI/FastDigest® XbaI (T↓CTAGA)
FspBI (BfaI)/FastDigest® BfaI (FspBI)
Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CTAGG)BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Eco130I (StyI)/FastDigest® StyI (Eco130I), FspBI (BfaI)/FastDigest® BfaI (FspBI), XmaJI (AvrII)/FastDigest® AvrII (XmaJI)
XmiI (AccI)/FastDigest® AccI (XmiI)* (GT↓ATAC)
CviAII (C↓ATG) FaiI
XmiI (AccI)/FastDigest® AccI (XmiI)* (GT↓CGAC)
BmeT110I* (CY↓CGAG), Bsp119I (BstBI)/FastDigest® Bsp119I (TT↓CGAA), Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I) (AT↓CGAT), TaqI/FastDigest® TaqI (T↓CGA)
TaqI/FastDigest® TaqI
202
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Recognition Sites Resulting from Fill-in of 5’-overhang and Self-ligation
Table 1.23. Newly generated recognition sites resulting from fill-in of 5’-overhang and self-ligation.
(continued on next page)
Restriction enzyme
Recognition sequence
Newly generated sequence after reaction
Restriction enzymes that cleave the newly generated recognition sequence
AbsI CC↓TCGAGG CCTCGATCGAGGBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, [MnlI/FastDigest® MnlI], PvuI/FastDigest® PvuI, [TaqI/FastDigest® TaqI]
Acc65I (Asp718I)/FastDigest® Acc65I G↓GTACC GGTACGTACC[Csp6I (CviQI)/FastDigest® Csp6I], Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), [RsaI/FastDigest® RsaI], SetI, TaiI (MaeII)/FastDigest® TaiI
AflIII* A↓CACGT ACACGCACGT [MaeII], [SetI], [TaiI (MaeII)/FastDigest® TaiI]
AflIII* A↓CATGT ACATGCATGT[CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDi-gest® NsiI (Mph1103I), [XceI (NspI)/FastDigest® NspI (XceI)]
AflIII* A↓CGCGT ACGCGCGCGT[Bsh1236I (BstUI)/FastDigest® Bsh1236I], Cac8I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDi-gest® HinP1I (Hin6I), MauBI/FastDigest® MauBI, PauI (BssHII)/FastDigest® BssHII (PteI)
AflIII* A↓CGTGT ACGTGCGTGT [MaeII], [SetI], [TaiI (MaeII)/FastDigest® TaiI]Alw44I (ApaLI)/FastDigest® ApaLI (Alw44I)
G↓TGCAC GTGCATGCACCac8I, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), [HpyCH4V], PaeI (SphI)/FastDi-gest® SphI (PaeI), XceI (NspI)/FastDigest® NspI (XceI)
BamHI/FastDigest® BamHI G↓GATCC GGATCGATCC[Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I)], [BspPI (AlwI)], Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I), [DpnI/FastDigest® DpnI], [MboI/FastDigest® MboI], TaqI/FastDigest® TaqI
BbvCI CC↓TCAGC CCTCATCAGC [MnlI/FastDigest® MnlI]
BclI/FastDigest® BclI T↓GATCA TGATCGATCA[Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I)], Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I), [DpnI/FastDigest® DpnI], [MboI/FastDigest® MboI], TaqI/FastDigest® TaqI
BcnI (NciI)/FastDigest® NciI (BcnI) CC↓SGG CCSSGG BseDI (BsaJI)/FastDigest® BsaJI (BseDI)
BcnI (NciI)/FastDigest® NciI (BcnI)* CC↓CGG CCCCGG[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], BseDI (BsaJI)/FastDigest® BsaJI (BseDI), [BssKI], [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]
BcnI (NciI)/FastDigest® NciI (BcnI)* CC↓GGG CCGGGG[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], BseDI (BsaJI)/FastDigest® BsaJI (BseDI), [BssKI], [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]
BcuI (SpeI)/FastDigest® SpeI (BcuI) A↓CTAGT ACTAGCTAGT AluI/FastDigest® AluI, CviJI, [FspBI (BfaI)/FastDigest® BfaI (FspBI)], SetIBfmI (SfcI)/FastDigest® SfcI (BfmI) C↓TRYAG CTRYATRYAG FaiIBfmI (SfcI)/FastDigest® SfcI (BfmI)* C↓TACAG CTACATACAG FaiIBfmI (SfcI)/FastDigest® SfcI (BfmI)* C↓TATAG CTATATATAG [FaiI]
BfmI (SfcI)/FastDigest® SfcI (BfmI)* C↓TGCAG CTGCATGCAGCac8I, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), [HpyCH4V], PaeI (SphI)/FastDi-gest® SphI (PaeI), XceI (NspI)/FastDigest® NspI (XceI)
BfmI (SfcI)/FastDigest® SfcI (BfmI)* C↓TGTAG CTGTATGTAG FaiI
BglII/FastDigest® BglII A↓GATCT AGATCGATCT[Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I)], Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I), [DpnI/FastDigest® DpnI], [MboI/FastDigest® MboI], TaqI/FastDigest® TaqI
Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)
CC↓NGG CCNNGG BseDI (BsaJI)/FastDigest® BsaJI (BseDI)
Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)*
CC↓AGG CCAAGG BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Eco130I (StyI)/FastDigest® StyI (Eco130I)
Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)*
CC↓CGG CCCCGG[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], BseDI (BsaJI)/FastDigest® BsaJI (BseDI), [BssKI], [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]
Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)*
CC↓GGG CCGGGG[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], BseDI (BsaJI)/FastDigest® BsaJI (BseDI), [BssKI], [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]
Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)*
CC↓TGG CCTTGG BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Eco130I (StyI)/FastDigest® StyI (Eco130I)
BmeT110I CY↓CGRG CYCGCGRG Bsh1236I (BstUI)/FastDigest® Bsh1236IBmgT120I GG↓NCC GGNNCC BspLI (NlaIV)/FastDigest® NlaIV (BspLI)BmgT120I* GG↓ACC GGAACC BspLI (NlaIV)/FastDigest® NlaIV (BspLI)
BmgT120I* GG↓CCC GGCCCC[BmgT120I], BspLI (NlaIV)/FastDigest® NlaIV (BspLI), [BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], [Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I)], [CviJI]
BmgT120I* GG↓GCC GGGGCC[BmgT120I], BspLI (NlaIV)/FastDigest® NlaIV (BspLI), [BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], [Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I)], [CviJI]
BmgT120I* GG↓TCC GGTTCC BspLI (NlaIV)/FastDigest® NlaIV (BspLI)Bpu10I/FastDigest® Bpu10I* CC↓TCAGC CCTCATCAGC [MnlI/FastDigest® MnlI]
BsaWI W↓CCGGW WCCGGCCGGWBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]
Note* Restriction enzymes that have degenerate recognition
sequences (i.e., recognize more than one sequence). Be aware that these restriction endonucleases will cleave sequences in addition to the one listed.
[ ] Enzymes that cleave the target both before and after the ligation.
• Fermentas FastDigest® enzymes are shown in ruby.• Fermentas conventional enzymes are shown in orange.
Single letter codeR = G or A; K = G or T; B = C, G or T;Y = C or T; S = C or G; D = A, G or T;W = A or T; H = A, C or T; N = G, A, T or C.M = A or C; V = A, C or G;
203
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.23. Newly generated recognition sites resulting from fill-in of 5’-overhang and self-ligation.
(continued on next page)
Restriction enzyme
Recognition sequence
Newly generated sequence after reaction
Restriction enzymes that cleave the newly generated recognition sequence
BseYI C↓CCAGC CCCAGCCAGC [BseYI], Cac8I, CviJIBshNI (BanI)/FastDigest® BanI (BshNI)* G↓GCACC GGCACGCACC Cac8I
BshNI (BanI)/FastDigest® BanI (BshNI)* G↓GCGCC GGCGCGCGCCBsh1236I (BstUI)/FastDigest® Bsh1236I, Cac8I, [HhaI/FastDigest® HhaI], [Hin6I (HinP1I)/Fast-Digest® HinP1I (Hin6I)], PauI (BssHII)/FastDigest® BssHII (PteI)
BshNI (BanI)/FastDigest® BanI (BshNI)* G↓GTACC GGTACGTACC[Csp6I (CviQI)/FastDigest® Csp6I], Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), [RsaI/FastDigest® RsaI], SetI, TaiI (MaeII)/FastDigest® TaiI
BshNI (BanI)/FastDigest® BanI (BshNI)* G↓GTGCC GGTGCGTGCC Cac8I
BshTI (AgeI)/FastDigest® AgeI (BshTI) A↓CCGGT ACCGGCCGGTBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), [Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)], CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]
Bsp119I (BstBI)/FastDigest® Bsp119I TT↓CGAA TTCGCGAA Bsh1236I (BstUI)/FastDigest® Bsh1236I, Hpy188III, Bsp68I (NruI)/FastDigest® NruI (RruI)
Bsp120I (PspOMI)/FastDigest® Bsp120I
G↓GGCCC GGGCCGGCCC[BmgT120I], [BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), [Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I)], [CviJI], FseI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI)
Bsp1407I (BsrGI)/FastDigest® Bsp1407I
T↓GTACA TGTACGTACA[Csp6I (CviQI)/FastDigest® Csp6I], Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), [RsaI/FastDigest® RsaI], SetI, TaiI (MaeII)/FastDigest® TaiI
Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I)
↓GATC GATCGATC[Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I)], Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I), [DpnI/FastDigest® DpnI], [MboI/FastDigest® MboI], TaqI/FastDigest® TaqI
BspTI (AflII)/FastDigest® AflII (BspTI) C↓TTAAG CTTAATTAAGPacI/FastDigest® PacI (TTAAT↓TAA), [FastDigest® MseI (SaqAI)], TasI (Tsp509I)/FastDigest® Tsp509I (TasI), [Tru1I (MseI)/FastDigest® Tru1I]
BssKI ↓CCNGG CCNGGCCNGG[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI
BssKI* ↓CCAGG CCAGGCCAGG[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI]
BssKI* ↓CCCGG CCCGGCCCGG[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], BmgT120I, [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI, [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]
BssKI* ↓CCGGG CCGGGCCGGG[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], BmgT120I, [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI, [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]
BssKI* ↓CCTGG CCTGGCCTGG[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI]
Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I) AT↓CGAT ATCGCGAT Bsh1236I (BstUI)/FastDigest® Bsh1236I, Hpy188III, Bsp68I (NruI)/FastDigest® NruI (RruI)
BtgI* C↓CATGG CCATGCATGG[CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDi-gest® NsiI (Mph1103I)
BtgI* C↓CGCGG CCGCGCGCGG[Bsh1236I (BstUI)/FastDigest® Bsh1236I], Cac8I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/Fast-Digest® HinP1I (Hin6I), MauBI/FastDigest® MauBI, [SsiI (AciI)/FastDigest® AciI (SsiI)], PauI (BssHII)/FastDigest® BssHII (PteI)
Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)
R↓CCGGY RCCGGCCGGYBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), [Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)], CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]
Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I)*
G↓GCCC GGCCGCCC[BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], [CviJI], SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), SsiI (AciI)/FastDigest® AciI (SsiI), TauI/FastDigest® TauI
Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I)*
G↓GGCC GGGCGGCC[BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], [CviJI], SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), TauI/FastDigest® TauI
Cfr9I (XmaI) C↓CCGGG CCCGGCCGGG
[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]
CfrI (EaeI) Y↓GGCCR YGGCCGGCCR[BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), [CfrI (EaeI)], [CviJI], FseI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI)
CpoI (RsrII)/FastDigest® RsrII (CpoI)* CG↓GACCG CGGACGACCG Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)CpoI (RsrII)/FastDigest® RsrII (CpoI)* CG↓GTCCG CGGTCGTCCG Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)
FastDigest® SexAI (CsiI) A↓CCWGGT ACCWGGCCWGGT[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI]
FastDigest® SexAI (CsiI)* A↓CCAGGT ACCAGGCCAGGT[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI], [SetI]
FastDigest® SexAI (CsiI)* A↓CCTGGT ACCTGGCCTGGT[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI], [SetI]
Csp6I (CviQI)/FastDigest® Csp6I G↓TAC GTATACBst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I), FaiI, Hpy8I (MjaIV)/FastDigest® Hpy8I, XmiI (AccI)/FastDigest® AccI (XmiI)
CviAII C↓ATG CATATG [FaiI], NdeI/FastDigest® NdeIEco130I (StyI)/FastDigest® StyI (Eco130I)*
C↓CATGG CCATGCATGG[CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDi-gest® NsiI (Mph1103I)
Eco130I (StyI)/FastDigest® StyI (Eco130I)*
C↓CTAGG CCTAGCTAGG AluI/FastDigest® AluI, CviJI, [FspBI (BfaI)/FastDigest® BfaI (FspBI)], SetI
Eco52I (EagI)/FastDigest® EagI (Eco52I)
C↓GGCCG CGGCCGGCCG
[Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)], [BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), [CfrI (EaeI)], [CviJI], [Eco52I (EagI)/FastDigest® EagI (Eco52I)], FseI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI)
204
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.23. Newly generated recognition sites resulting from fill-in of 5’-overhang and self-ligation.
(continued on next page)
Restriction enzyme
Recognition sequence
Newly generated sequence after reaction
Restriction enzymes that cleave the newly generated recognition sequence
Eco81I (Bsu36I)/FastDigest® Bsu36I (Eco81I)*
CC↓TCAGG CCTCATCAGG [MnlI/FastDigest® MnlI]
Eco88I (AvaI)/FastDigest® AvaI (Eco88I)
C↓YCGRG CYCGRYCGRG Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)
Eco88I (AvaI)/FastDigest® AvaI (Eco88I)*
C↓CCGAG CCCGACCGAG Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)
Eco88I (AvaI)/FastDigest® AvaI (Eco88I)*
C↓CCGGG CCCGGCCGGG
[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]
Eco88I (AvaI)/FastDigest® AvaI (Eco88I)*
C↓TCGAG CTCGATCGAGBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PvuI/FastDigest® PvuI, [TaqI/FastDigest® TaqI]
Eco88I (AvaI)/FastDigest® AvaI (Eco88I)*
C↓TCGGG CTCGGTCGGG Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)
Eco91I (BstEII)/FastDigest® Eco91I G↓GTNACC GGTNACGTNACC MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest® TaiIEco91I (BstEII)/FastDigest® Eco91I* G↓GTAACC GGTAACGTAACC MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest® TaiIEco91I (BstEII)/FastDigest® Eco91I* G↓GTCACC GGTCACGTCACC AjiI (BmgBI), MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest® NmuCI], SetI, TaiI (MaeII)/FastDigest® TaiIEco91I (BstEII)/FastDigest® Eco91I* G↓GTGACC GGTGACGTGACC [HphI], MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest® NmuCI], SetI, TaiI (MaeII)/FastDigest® TaiIEco91I (BstEII)/FastDigest® Eco91I* G↓GTTACC GGTTACGTTACC MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest® TaiIEcoO109I (DraII)/FastDigest® EcoO109I*
RG↓GCCCY RGGCCGCCCY[BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], [CviJI], SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), SsiI (AciI)/FastDigest® AciI (SsiI), TauI/FastDigest® TauI
EcoO109I (DraII)/FastDigest® EcoO109I*
RG↓GGCCY RGGGCGGCCY[BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], [CviJI], SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), TauI/FastDigest® TauI
EcoRI/FastDigest® EcoRI G↓AATTC GAATTAATTCPdmI (XmnI)/FastDigest® PdmI, FastDigest® MseI (SaqAI), [TasI (Tsp509I)/FastDigest® Tsp509I (TasI)], Tru1I (MseI)/FastDigest® Tru1I, VspI (AseI)/FastDigest® AseI (VspI)
EcoRII ↓CCWGG CCWGGCCWGG[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI]
EcoRII* ↓CCAGG CCAGGCCAGG[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI]
EcoRII* ↓CCTGG CCTGGCCTGG[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI]
FatI ↓CATG CATGCATG[CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDi-gest® NsiI (Mph1103I)
FspBI (BfaI)/FastDigest® BfaI (FspBI) C↓TAG CTATAG BfmI (SfcI)/FastDigest® SfcI (BfmI), FaiIHin1I (BsaHI)/FastDigest® BsaHI (Hin1I) GR↓CGYC GRCGCGYC Bsh1236I (BstUI)/FastDigest® Bsh1236IHin6I (HinP1I)/FastDigest® HinP1I (Hin6I)
G↓CGC GCGCGCBsh1236I (BstUI)/FastDigest® Bsh1236I, Cac8I, [HhaI/FastDigest® HhaI], [Hin6I (HinP1I)/Fast-Digest® HinP1I (Hin6I)], PauI (BssHII)/FastDigest® BssHII (PteI)
HindIII/FastDigest® HindIII A↓AGCTT AAGCTAGCTT[AluI/FastDigest® AluI], BspOI (BmtI)/FastDigest® BmtI (BspOI), Cac8I, [CviJI], FspBI (BfaI)/FastDigest® BfaI (FspBI), NheI/FastDigest® NheI, [SetI]
HpaII/FastDigest® HpaII C↓CGG CCGCGGBseDI (BsaJI)/FastDigest® BsaJI (BseDI), Bsh1236I (BstUI)/FastDigest® Bsh1236I, BtgI, Cfr42I (SacII), MspA1I, SsiI (AciI)/FastDigest® AciI (SsiI)
Kpn2I (BspEI)/FastDigest® Kpn2I T↓CCGGA TCCGGCCGGABsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]
MaeII A↓CGT ACGCGT AflIII, Bsh1236I (BstUI)/FastDigest® Bsh1236I, MluI/FastDigest® MluIMaeIII ↓GTNAC GTNACGTNAC MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest® TaiIMaeIII* ↓GTAAC GTAACGTAAC MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest® TaiIMaeIII* ↓GTCAC GTCACGTCAC AjiI (BmgBI), MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest® NmuCI], SetI, TaiI (MaeII)/FastDigest® TaiIMaeIII* ↓GTGAC GTGACGTGAC MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest® NmuCI], SetI, TaiI (MaeII)/FastDigest® TaiIMaeIII* ↓GTTAC GTTACGTTAC MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest® TaiI
MauBI/FastDigest® MauBI CG↓CGCGCG CGCGCGCGCGCG[Bsh1236I (BstUI)/FastDigest® Bsh1236I], [Cac8I], [HhaI/FastDigest® HhaI], [Hin6I (HinP1I)/Fast-Digest® HinP1I (Hin6I)], [MauBI/FastDigest® MauBI], [PauI (BssHII)/FastDigest® BssHII (PteI)]
MboI/FastDigest® MboI ↓GATC GATCGATC[Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I)], Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I), [DpnI/FastDigest® DpnI], [MboI/FastDigest® MboI], TaqI/FastDigest® TaqI
MluI/FastDigest® MluI A↓CGCGT ACGCGCGCGT[Bsh1236I (BstUI)/FastDigest® Bsh1236I], Cac8I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDi-gest® HinP1I (Hin6I), MauBI/FastDigest® MauBI, PauI (BssHII)/FastDigest® BssHII (PteI)
MreI (Sse232I)/FastDigest® MreI CG↓CCGGCG CGCCGGCCGGCG
Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), [Cac8I], [Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)], CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI], [NgoMIV], [PdiI (NaeI)/FastDigest® NaeI (PdiI)]
FastDigest® MseI (SaqAI) T↓TAA TTATAA AanI (PsiI)/FastDigest® PsiI (AanI), FaiI
MspI (HpaII)/FastDigest® MspI C↓CGG CCGCGGBseDI (BsaJI)/FastDigest® BsaJI (BseDI), Bsh1236I (BstUI)/FastDigest® Bsh1236I, BtgI, Cfr42I (SacII), MspA1I, SsiI (AciI)/FastDigest® AciI (SsiI)
MunI (MfeI)/FastDigest® MfeI (MunI) C↓AATTG CAATTAATTGFastDigest® MseI (SaqAI), [TasI (Tsp509I)/FastDigest® Tsp509I (TasI)], Tru1I (MseI)/FastDigest® Tru1I, VspI (AseI)/FastDigest® AseI (VspI)
MvaI (BstNI)/FastDigest® MvaI CC↓WGG CCWWGG BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Eco130I (StyI)/FastDigest® StyI (Eco130I)MvaI (BstNI)/FastDigest® MvaI* CC↓AGG CCAAGG BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Eco130I (StyI)/FastDigest® StyI (Eco130I)MvaI (BstNI)/FastDigest® MvaI* CC↓TGG CCTTGG BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Eco130I (StyI)/FastDigest® StyI (Eco130I)
205
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.23. Newly generated recognition sites resulting from fill-in of 5’-overhang and self-ligation.
(continued on next page)
Restriction enzyme
Recognition sequence
Newly generated sequence after reaction
Restriction enzymes that cleave the newly generated recognition sequence
NarI GG↓CGCC GGCGCGCCBsh1236I (BstUI)/FastDigest® Bsh1236I, Cac8I, [HhaI/FastDigest® HhaI], [Hin6I (HinP1I)/FastDi-gest® HinP1I (Hin6I)], SgsI (AscI)/FastDigest® AscI (SgsI), PauI (BssHII)/FastDigest® BssHII (PteI)
NcoI/FastDigest® NcoI C↓CATGG CCATGCATGG[CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDi-gest® NsiI (Mph1103I)
NdeI/FastDigest® NdeI CA↓TATG CATATATG [FaiI]
NgoMIV G↓CCGGC GCCGGCCGGC
Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), [Cac8I], [Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)], CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI], [NgoMIV], [PdiI (NaeI)/FastDigest® NaeI (PdiI)]
NheI/FastDigest® NheI G↓CTAGC GCTAGCTAGCAluI/FastDigest® AluI, [BspOI (BmtI)/FastDigest® BmtI (BspOI)], [Cac8I], CviJI, [FspBI (BfaI)/FastDigest® BfaI (FspBI)], [NheI/FastDigest® NheI], SetI
NmuCI (Tsp45I)/FastDigest® NmuCI ↓GTSAC GTSACGTSAC MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest® NmuCI], SetI, TaiI (MaeII)/FastDigest® TaiINmuCI (Tsp45I)/FastDigest® NmuCI* ↓GTCAC GTCACGTCAC AjiI (BmgBI), MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest® NmuCI], SetI, TaiI (MaeII)/FastDigest® TaiINmuCI (Tsp45I)/FastDigest® NmuCI* ↓GTGAC GTGACGTGAC MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest® NmuCI], SetI, TaiI (MaeII)/FastDigest® TaiI
NotI/FastDigest® NotI GC↓GGCCGC GCGGCCGGCCGC
[Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)], [BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), [CfrI (EaeI)], [CviJI], [Eco52I (EagI)/FastDigest® EagI (Eco52I)], FseI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI), [SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI)], [SsiI (AciI)/FastDigest® AciI (SsiI)], [TauI/FastDigest® TauI]
PagI (BspHI)/FastDigest® BspHI (PagI) T↓CATGA TCATGCATGA[CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDi-gest® NsiI (Mph1103I)
PasI* CC↓CAGGG CCCAGCAGGG BseYI, EcoP15I
PauI (BssHII)/FastDigest® BssHII (PteI) G↓CGCGC GCGCGCGCGC[Bsh1236I (BstUI)/FastDigest® Bsh1236I], [Cac8I], [HhaI/FastDigest® HhaI], [Hin6I (HinP1I)/Fast-Digest® HinP1I (Hin6I)], MauBI/FastDigest® MauBI, [PauI (BssHII)/FastDigest® BssHII (PteI)]
Pfl23II (BsiWI)/FastDigest® BsiWI (Pfl23II)
C↓GTACG CGTACGTACG[Csp6I (CviQI)/FastDigest® Csp6I], Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I), MaeII, [Pfl23II (BsiWI)/FastDigest® BsiWI (Pfl23II)], Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), [RsaI/FastDi-gest® RsaI], SetI, TaiI (MaeII)/FastDigest® TaiI
PfoI/FastDigest® PfoI T↓CCNGGA TCCNGGCCNGGA[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI
PfoI/FastDigest® PfoI* T↓CCAGGA TCCAGGCCAGGA[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI]
PfoI/FastDigest® PfoI* T↓CCCGGA TCCCGGCCCGGA[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], BmgT120I, [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI, [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]
PfoI/FastDigest® PfoI* T↓CCGGGA TCCGGGCCGGGA[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], BmgT120I, [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI, [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]
PfoI/FastDigest® PfoI* T↓CCTGGA TCCTGGCCTGGA[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI]
PscI (PciI) A↓CATGT ACATGCATGT[CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDi-gest® NsiI (Mph1103I), [XceI (NspI)/FastDigest® NspI (XceI)]
Psp1406I (AclI)/FastDigest® AclI (Psp1406I)
AA↓CGTT AACGCGTT AflIII, Bsh1236I (BstUI)/FastDigest® Bsh1236I, MluI/FastDigest® MluI
PspXI VC↓TCGAGB VCTCGATCGAGBBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PvuI/FastDigest® PvuI, [TaqI/FastDigest® TaqI]
PsuI (BstYI)/FastDigest® PsuI R↓GATCY RGATCGATCY[Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I)], Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I), [DpnI/FastDigest® DpnI], [MboI/FastDigest® MboI], TaqI/FastDigest® TaqI
PsyI (Tth111I)/FastDigest® PsyI GACN↓NNGTC GACNNNNGTC BoxI (PshAI)/FastDigest® PshAI (BoxI)PsyI (Tth111I)/FastDigest® PsyI* GACN↓ANGTC GACNAANGTC BoxI (PshAI)/FastDigest® PshAI (BoxI)PsyI (Tth111I)/FastDigest® PsyI* GACN↓CNGTC GACNCCNGTC BoxI (PshAI)/FastDigest® PshAI (BoxI)PsyI (Tth111I)/FastDigest® PsyI* GACN↓GNGTC GACNGGNGTC BoxI (PshAI)/FastDigest® PshAI (BoxI)PsyI (Tth111I)/FastDigest® PsyI* GACN↓TNGTC GACNTTNGTC BoxI (PshAI)/FastDigest® PshAI (BoxI)
SalI/FastDigest® SalI G↓TCGAC GTCGATCGACBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PvuI/FastDigest® PvuI, [TaqI/FastDigest® TaqI]
FastDigest® SanDI (KflI)* GG↓GACCC GGGACGACCC [FaqI (BsmFI)/FastDigest® BsmFI (FaqI)]SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI) GC↓NGC GCNNGC Cac8ISatI (Fnu4HI)/FastDigest® Fnu4HI (SatI)*
GC↓AGC GCAAGC Cac8I
SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI)*
GC↓CGC GCCCGC Cac8I, SmuI (FauI), [SsiI (AciI)/FastDigest® AciI (SsiI)]
SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI)*
GC↓GGC GCGGGC Cac8I
SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI)*
GC↓TGC GCTTGC Cac8I
SgrAI CR↓CCGGYG CRCCGGCCGGYGBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), [Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)], CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]
206
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.23. Newly generated recognition sites resulting from fill-in of 5’-overhang and self-ligation.
Restriction enzyme
Recognition sequence
Newly generated sequence after reaction
Restriction enzymes that cleave the newly generated recognition sequence
SgrDI CG↓TCGACG CGTCGATCGACGBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, [Hpy99I], MboI/FastDigest® MboI, PvuI/FastDigest® PvuI, [TaqI/FastDigest® TaqI]
SgsI (AscI)/FastDigest® AscI (SgsI) GG↓CGCGCC GGCGCGCGCGCC[Bsh1236I (BstUI)/FastDigest® Bsh1236I], [Cac8I], [HhaI/FastDigest® HhaI], [Hin6I (HinP1I)/Fast-Digest® HinP1I (Hin6I)], MauBI/FastDigest® MauBI, [PauI (BssHII)/FastDigest® BssHII (PteI)]
SmoI (SmlI)* C↓TCGAG CTCGATCGAGBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PvuI/FastDigest® PvuI, [TaqI/FastDigest® TaqI]
SmoI (SmlI)* C↓TTAAG CTTAATTAAGPacI/FastDigest® PacI (TTAAT↓TAA), [FastDigest® MseI (SaqAI)], TasI (Tsp509I)/FastDigest® Tsp509I (TasI), [Tru1I (MseI)/FastDigest® Tru1I]
SsiI (AciI)/FastDigest® AciI (SsiI) C↓CGC CCGCGCBsh1236I (BstUI)/FastDigest® Bsh1236I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), [SsiI (AciI)/FastDigest® AciI (SsiI)]
SspDI (KasI) G↓GCGCC GGCGCGCGCCBsh1236I (BstUI)/FastDigest® Bsh1236I, Cac8I, [HhaI/FastDigest® HhaI], [Hin6I (HinP1I)/Fast-Digest® HinP1I (Hin6I)], PauI (BssHII)/FastDigest® BssHII (PteI)
TaqI/FastDigest® TaqI T↓CGA TCGCGA Bsh1236I (BstUI)/FastDigest® Bsh1236I, Hpy188III, Bsp68I (NruI)/FastDigest® NruI (RruI)TasI (Tsp509I)/FastDigest® Tsp509I (TasI)
↓AATT AATTAATTFastDigest® MseI (SaqAI), [TasI (Tsp509I)/FastDigest® Tsp509I (TasI)], Tru1I (MseI)/FastDigest® Tru1I, VspI (AseI)/FastDigest® AseI (VspI)
TatI/FastDigest® TatI W↓GTACW WGTACGTACW[Csp6I (CviQI)/FastDigest® Csp6I], Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), [RsaI/FastDigest® RsaI], SetI, TaiI (MaeII)/FastDigest® TaiI
Tru1I (MseI)/FastDigest® Tru1I T↓TAA TTATAA AanI (PsiI)/FastDigest® PsiI (AanI), FaiITseI G↓CWGC GCWGCWGC [SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI)], [TseI]
TseI* G↓CAGC GCAGCAGC[BseXI (BbvI)/FastDigest® BseXI], EcoP15I, [Lsp1109I (BbvI)/FastDigest® BbvI (Lsp1109I)], [SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI)], [TseI]
TseI* G↓CTGC GCTGCTGC [SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI)], [TseI]VspI (AseI)/FastDigest® AseI (VspI) AT↓TAAT ATTATAAT AanI (PsiI)/FastDigest® PsiI (AanI), FaiI
XapI (ApoI)/FastDigest® XapI R↓AATTY RAATTAATTYFastDigest® MseI (SaqAI), [TasI (Tsp509I)/FastDigest® Tsp509I (TasI)], Tru1I (MseI)/FastDigest® Tru1I, VspI (AseI)/FastDigest® AseI (VspI)
XbaI/FastDigest® XbaI T↓CTAGA TCTAGCTAGA AluI/FastDigest® AluI, CviJI, [FspBI (BfaI)/FastDigest® BfaI (FspBI)], SetI
XhoI/FastDigest® XhoI C↓TCGAG CTCGATCGAGBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PvuI/FastDigest® PvuI, [TaqI/FastDigest® TaqI]
XmaJI (AvrII)/FastDigest® AvrII (XmaJI) C↓CTAGG CCTAGCTAGG AluI/FastDigest® AluI, CviJI, [FspBI (BfaI)/FastDigest® BfaI (FspBI)], SetIXmiI (AccI)/FastDigest® AccI (XmiI)* GT↓ATAC GTATATAC [FaiI]XmiI (AccI)/FastDigest® AccI (XmiI)* GT↓CGAC GTCGCGAC Bsh1236I (BstUI)/FastDigest® Bsh1236I, Hpy188III, Bsp68I (NruI)/FastDigest® NruI (RruI)XmiI (AccI)/FastDigest® AccI (XmiI)* GT↓CTAC GTCTCTAC Alw26I (BsmAI)/FastDigest® Alw26I
Table 1.24. Newly generated recognition sites resulting from removal of 3’-overhang and self-ligation.
Recognition Sites Resulting from Removal of 3’-overhang and Self-ligation
Restriction enzyme
Recognition sequence
Newly generated sequence after reaction
Restriction enzymes that cleave the newly generated recognition sequence
AasI (DrdI)/FastDigest® DrdI (AasI) GACNNNN↓NNGTC GACNNNNGTC BoxI (PshAI)/FastDigest® PshAI (BoxI)AdeI (DraIII)/FastDigest® DraIII (AdeI)
CACNNN↓GTG CACGTGEco72I (PmlI)/FastDigest® PmlI (Eco72I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI
AgsI TTS↓AA TTAA FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1IBglI/FastDigest® BglI GCCNNNN↓NGGC GCCNNGGC BseDI (BsaJI)/FastDigest® BsaJI (BseDI)Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)
CGRY↓CG CGCG Bsh1236I (BstUI)/FastDigest® Bsh1236I
CaiI (AlwNI)/FastDigest® AlwNI (CaiI)
CAGNNN↓CTG CAGCTG AluI/FastDigest® AluI, CviJI, MspA1I, PvuII/FastDigest® PvuII, SetI
Cfr42I (SacII) CCGC↓GG CCGG HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIEam1105I (AhdI)/FastDigest® Eam1105I
GACNNN↓NNGTC GACNNNNGTC BoxI (PshAI)/FastDigest® PshAI (BoxI)
FseI GGCCGG↓CC GGCC [BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], [CviJI]Hpy188I TCN↓GA TCGA TaqI/FastDigest® TaqIPacI/FastDigest® PacI (TTAAT↓TAA)
TTAAT↓TAA TTATAA AanI (PsiI)/FastDigest® PsiI (AanI), FaiI
PvuI/FastDigest® PvuI CGAT↓CG CGCG Bsh1236I (BstUI)/FastDigest® Bsh1236ISdaI (SbfI)/FastDigest® SbfI (SdaI)
CCTGCA↓GG CCGG HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI
SfaAI (AsiSI)/FastDigest® AsiSI (SfaAI)
GCGAT↓CGC GCGCGCBsh1236I (BstUI)/FastDigest® Bsh1236I, Cac8I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/Fast-Digest® HinP1I (Hin6I), PauI (BssHII)/FastDigest® BssHII (PteI)
SfiI/FastDigest® SfiI GGCCNNNN↓NGGCC GGCCNNGGCC BseDI (BsaJI)/FastDigest® BsaJI (BseDI), [BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], [CviJI]TaaI (HpyCH4III)/FastDigest® TaaI ACN↓GT ACGT MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI
Note• Fermentas FastDigest® enzymes are shown in ruby.• Fermentas conventional enzymes are shown in orange.
Single letter codeR = G or A; K = G or T; B = C, G or T; M = A or CY = C or T; S = C or G; D = A, G or T; V = A, C or G;W = A or T; H = A, C or T; N = G, A, T or C.
207
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Selection GuidesAlphabetic List of Commercially Available Restriction Enzymes
Table 1.25. Commercially available restriction enzymes.
(continued on next page)
Single letter codeR = G or A; H = A, C or T; Y = C or T; V = A, C or G;W = A or T; B = C, G or T;M = A or C; D = A, G or T;K = G or T; N = G, A, T or C.S = C or G;
Note[ ] Enzymes have different cleavage specificities (neoschizomers).m Isoschizomers with different sensitivity to methylation.* Restriction enzymes produced by Fermentas are shown in bold.• Fermentas FastDigest® enzymes are shown in ruby.• Fermentas Conventional enzymes are shown in orange.
Enzyme Specificity 5’→3’ FastDigest® enzyme Page Conventional enzyme PageCommercially available isos-chizomers from other vendors
AanI TTA↓TAA FastDigest® PsiI (AanI) 55 AanI (PsiI) 80 PsiIAarI CACCTGC(4/8)↓ AarI 80AasI GACNNNN↓NNGTC FastDigest® DrdI (AasI) 34 AasI (DrdI) 80 DrdI, DseDIAatI AGG↓CCT FastDigest® StuI (Eco147I) 64 Eco147I (StuI) 115 PceI, SseBI, StuIAatII GACGT↓C FastDigest® AatII 11 AatII 81 [ZraIm]AbsI CC↓TCGAGGAcc16I TGC↓GCA FastDigest® FspI (NsbI) 39 NsbI (FspI) 133 AviII, FspIAcc36I ACCTGC(4/8)↓ FastDigest® BspMI (BveI) 29 BveI (BspMI) 104 BfuAI, BspMI
Acc65I G↓GTACCFastDigest® Acc65I, [FastDigest® KpnIm](GGTAC↓C)
1143
Acc65I (Asp718I), [KpnIm](GGTAC↓C)
81124
Asp718Im
AccB1I G↓GYRCC FastDigest® BanI (BshNI) 19 BshNI (BanI) 97 BanI, BspT107IAccB7I CCANNNN↓NTGG FastDigest® PflMI (Van91I) 54 Van91I (PflMI) 154 BasI, PflMIAccBSI CCGCTC(-3/-3)↓ FastDigest® BsrBI (MbiI) 30 MbiI (BsrBI) 126 BsrBIAccI GT↓MKAC FastDigest® AccI (XmiI) 11 XmiI (AccI) 157 AccI, FblIAccII CG↓CG FastDigest® Bsh1236I 25 Bsh1236I (BstUI) 97 BspFNI, BstFNI, BstUI, MvnIAccIII T↓CCGGA FastDigest® Kpn2Im 44 Kpn2I (BspEI)m 124 Aor13HI, BseAIm, Bsp13I, BspEIm, MroIm
AciI CCGC(-3/-1)↓ FastDigest® AciI (SsiI) 12 SsiI (AciI) 149 AciI, BspACIAclI AA↓CGTT FastDigest® AclI (Psp1406I) 12 Psp1406I (AclI) 139 AclIAclWI GGATC(4/5)↓ BspPI (AlwI) 101 AlwIAcoI Y↓GGCCR CfrI (EaeI) 105 EaeIAcsI R↓AATTY FastDigest® XapI 68 XapI (ApoI) 155 ApoIAcuI CTGAAG(16/14)↓ FastDigest® AcuI (Eco57I) 12 Eco57I (AcuI) 112 AcuIAcvI CAC↓GTG FastDigest® PmlI (Eco72I) 54 Eco72I (PmlI) 113 BbrPI, PmaCI, PmlI, PspCIAcyI GR↓CGYC FastDigest® BsaHI (Hin1I) 24 Hin1I (BsaHI) 120 BsaHI, BssNI, BstACI, Hsp92IAdeI CACNNN↓GTG FastDigest® DraIII (AdeI) 34 AdeI (DraIII) 82 DraIII
AfaI GT↓AC[FastDigest® Csp6Im](G↓TAC), FastDigest® RsaI
3258
[Csp6I (CviQI)m](G↓TAC), RsaI
107141
[CviQIm], [RsaNI]
AfeI AGC↓GCT FastDigest® AfeI (Eco47III) 13 Eco47III (AfeI) 111 AfeI, Aor51HIAfiI CCNNNNN↓NNGG FastDigest® BslI (BseLI) 26 BseLI (BslI) 95 Bsc4I, BslIAflII C↓TTAAG FastDigest® AflII (BspTI) 13 BspTI (AflII) 101 AflII, BfrI, Bst98I, BstAFI, MspCI, Vha464IAflIII A↓CRYGTAgeI A↓CCGGT FastDigest® AgeI (BshTI) 13 BshTI (AgeI) 98 AgeI, AsiGI, CspAIm, PinAIAgsI TTS↓AAAhdI GACNNN↓NNGTC FastDigest® Eam1105I 35 Eam1105I (AhdI) 109 AspEI, BmeRI, DriI, EclHKIAhlI A↓CTAGT FastDigest® SpeI (BcuI) 63 BcuI (SpeI) 88 SpeIAjiI CACGTC(-3/-3)↓ AjiI (BmgBI) 82 BmgBI, BtrI
AjnI ↓CCWGG [FastDigest® MvaI](CC↓WGG) 48EcoRIIm, [MvaI (BstNI)](CC↓WGG)
116130
[BseBI], [Bst2UI], [BstNI], [BstOI], Psp6I, PspGIm
AjuI ↓(6/11)CCAA(N)7TTC(12/7)↓ FastDigest® AjuI 14 AjuI 83AleI CACNN↓NNGTG FastDigest® AleI (OliI) 14 OliI (AleI) 134 AleIAlfI ↓(10/12)GCA(N)6TGC(12/10)↓ AlfI 83AloI ↓(7/12)GAAC(N)6TCC(12/7)↓ AloI 84AluI AG↓CT FastDigest® AluI 14 AluI 84 AluBIAluBI AG↓CT FastDigest® AluI 14 AluI 84Alw21I GWGCW↓C FastDigest® Alw21I 15 Alw21I (BsiHKAI) 84 Bbv12I, BsiHKAIAlw26I GTCTC(1/5)↓ FastDigest® Alw26I 15 Alw26I (BsmAI) 85 BsmAIm, BstMAIAlw44I G↓TGCAC FastDigest® ApaLI (Alw44I) 16 Alw44I (ApaLI) 85 ApaLIm, VneIAlwI GGATC(4/5)↓ BspPI (AlwI) 101 AclWIAlwNI CAGNNN↓CTG FastDigest® AlwNI (CaiI) 15 CaiI (AlwNI) 104 AlwNIAma87I C↓YCGRG FastDigest® AvaI (Eco88I) 17 Eco88I (AvaI) 113 AvaI, [BmeT110I], BsiHKCI, BsoBIAor13HI T↓CCGGA FastDigest® Kpn2I 44 Kpn2I (BspEI) 124 AccIII, BseAI, Bsp13I, BspEI, MroIAor51HI AGC↓GCT FastDigest® AfeI (Eco47III) 13 Eco47III (AfeI) 111 AfeI
ApaI GGGCC↓CFastDigest® ApaI, [FastDigest® Bsp120I](G↓GGCCC)
1628
ApaI, [Bsp120I (PspOMI)](G↓GGCCC)
8599
[PspOMI]
208
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.25. Commercially available restriction enzymes.
(continued on next page)
Enzyme Specificity 5’→3’ FastDigest® enzyme Page Conventional enzyme PageCommercially available isos-chizomers from other vendors
ApaLI G↓TGCAC FastDigest® ApaLI (Alw44I)m 16 Alw44I (ApaLI)m 85 ApaLI, VneIApeKI G↓CWGC TseIApoI R↓AATTY FastDigest® XapI 68 XapI (ApoI) 155 AcsIArsI ↓(8/13)GAC(N)6TTYG(11/6)↓AscI GG↓CGCGCC FastDigest® AscI (SgsI) 16 SgsI (AscI) 147 AscI, PalAIAseI AT↓TAAT FastDigest® AseI (VspI) 17 VspI (AseI) 154 AseI, PshBIAsiGI A↓CCGGT FastDigest® AgeI (BshTI) 13 BshTI (AgeI) 98 AgeI, CspAI, PinAIAsiSI GCGAT↓CGC FastDigest® AsiSI (SfaAI) 17 SfaAI (AsiSI) 145 AsiSI, RgaI, SgfIAsp700I GAANN↓NNTTC FastDigest® PdmI 53 PdmI (XmnI) 136 MroXI, XmnI
Asp718I G↓GTACCFastDigest® Acc65Im, [FastDigest® KpnIm](GGTAC↓C)
1143
Acc65I (Asp718I)m, [KpnIm](GGTAC↓C)
81124
AspA2I C↓CTAGG FastDigest® AvrII (XmaJI) 18 XmaJI (AvrII) 156 AvrII, BlnIAspEI GACNNN↓NNGTC FastDigest® Eam1105I 35 Eam1105I (AhdI) 109 AhdI, BmeRI, DriI, EclHKIAspI GACN↓NNGTC FastDigest® PsyI 57 PsyI (Tth111I) 140 PflFI, Tth111I
AspLEI GCG↓CFastDigest® HhaI, [FastDigest® HinP1I (Hin6I)](G↓CGC)
4042
HhaI, [Hin6I (HinP1I)](G↓CGC)
120121
BstHHI, CfoI, [HinP1I], [HspAI]
AspS9I G↓GNCC FastDigest® Sau96I (Cfr13I) 60 Cfr13I (Sau96I) 106 [BmgT120I], PspPI, Sau96IAssI AGT↓ACT FastDigest® ScaI 61 ScaI 144 BmcAI, ZrmIAsuC2I CC↓SGG FastDigest® NciI (BcnI) 49 BcnI (NciI) 88 BpuMI, NciIAsuHPI GGTGA(8/7)↓ HphI 123
AsuII TT↓CGAA FastDigest® Bsp119Im 27 Bsp119I (BstBI)m 99Bpu14I, BspT104Im, BstBIm, Csp45I, NspVm, SfuI
AsuNHI G↓CTAGC[FastDigest® BmtI (BspOI)](GCTAG↓C), FastDigest® NheI
2250
[BspOI (BmtI)](GCTAG↓C), NheI
100132
[BmtI]
AvaI C↓YCGRG FastDigest® AvaI (Eco88I)m 17 Eco88I (AvaI)m 113Ama87I, AvaI, [BmeT110Im], BsiHKCI, BsoBIm
AvaII G↓GWCC FastDigest® AvaII (Eco47I) 18 Eco47I (AvaII) 111 AvaII, Bme18I, SinI, VpaK11BIAviII TGC↓GCA FastDigest® FspI (NsbI) 39 NsbI (FspI) 133 Acc16I, FspIAvrII C↓CTAGG FastDigest® AvrII (XmaJI) 18 XmaJI (AvrII) 156 AspA2I, AvrII, BlnIAxyI CC↓TNAGG FastDigest® Bsu36I (Eco81I) 32 Eco81I (Bsu36I) 113 Bse21I, Bsu36IBaeGI GKGCM↓C FastDigest® Bme1580I (BseSI) 21 BseSI (Bme1580I) 96 BstSLIBaeI ↓(10/15)AC(N)4GTAYC(12/7)↓BalI TGG↓CCA FastDigest® MscI (MlsI) 47 MlsI (MscI) 128 MluNI, MscI, Msp20IBamHI G↓GATCC FastDigest® BamHI 18 BamHI 86BanI G↓GYRCC FastDigest® BanI (BshNI) 19 BshNI (BanI) 97 AccB1I, BanI, BspT107IBanII GRGCY↓C Eco24I (BanII) 110 EcoT38I, FriOI
BanIII AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103Bsa29I, BseCI, BshVI, BspDI, BspXI, BsuTUI, ClaI
BarI ↓(7/12)GAAG(N)6TAC(12/7)↓BasI CCANNNN↓NTGG FastDigest® PflMI (Van91I) 54 Van91I (PflMI) 154 AccB7I, PflMIBauI CACGAG(-5/-1)↓ BauI (BssSI) 87 BssSI, Bst2BI
BbeI GGCGC↓C [FastDigest® EheI](GGC↓GCC) 38[EheI (SfoI)](GGC↓GCC), [SspDI (KasI)](G↓GCGCC)
117150
[DinI], [EgeI], [KasI], [Mly113I], [NarI], [SfoIm]
BbrPI CAC↓GTG FastDigest® PmlI (Eco72I) 54 Eco72I (PmlI) 113 AcvI, PmaCI, PmlI, PspCIBbsI GAAGAC(2/6)↓ FastDigest® BbsI (BpiI) 19 BpiI (BbsI) 92 BbsI, BpuAI, BstV2IBbuI GCATG↓C FastDigest® SphI (PaeI) 64 PaeI (SphI) 134 SphIBbv12I GWGCW↓C FastDigest® Alw21I 15 Alw21I (BsiHKAI) 84 BsiHKAIBbvCI CCTCAGC(-5/-2)↓
BbvI GCAGC(8/12)↓ FastDigest® BbvI (Lsp1109I), FastDigest® BseXI
1925
Lsp1109I (BbvI), BseXI (BbvI)
12596
BbvI, BstV1I
BccI CCATC(4/5)↓BceAI ACGGC(12/14)↓BcgI ↓(10/12)CGA(N)6TGC(12/10)↓BciVI GTATCC(6/5)↓ BfuI (BciVI) 90BclI T↓GATCA FastDigest® BclI 20 BclI 87 FbaI, Ksp22IBcnI CC↓SGG FastDigest® NciI (BcnI) 49 BcnI (NciI) 88 AsuC2I, BpuMI, NciIBcuI A↓CTAGT FastDigest® SpeI (BcuI) 63 BcuI (SpeI) 88 AhlI, SpeIBdaI ↓(10/12)TGA(N)6TCA(12/10)↓ BdaI 88BfaI C↓TAG FastDigest® BfaI (FspBI) 20 FspBI (BfaI) 119 BfaI, MaeI, XspIBfiI ACTGGG(5/4)↓ BfiI (BmrI) 89 BmrI, BmuIBfmI C↓TRYAG FastDigest® SfcI (BfmI) 62 BfmI (SfcI) 89 BstSFI, SfcIBfrI C↓TTAAG FastDigest® AflII (BspTI) 13 BspTI (AflII) 101 AflII, Bst98I, BstAFI, MspCI, Vha464IBfuAI ACCTGC(4/8)↓ FastDigest® BspMI (BveI) 29 BveI (BspMI) 104 Acc36I, BspMI
BfuCI ↓GATCFastDigest® Sau3AI (Bsp143I), FastDigest® MboIm
6044
Bsp143I (Sau3AI), MboIm
99127
BssMI, [BstKTIm], BstMBI, DpnIIm, Kzo9I, NdeIIm, Sau3AIm
BfuI GTATCC(6/5)↓ BfuI (BciVI) 90 BciVIBglI GCCNNNN↓NGGC FastDigest® BglI 20 BglI 90
209
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.25. Commercially available restriction enzymes.
(continued on next page)
Enzyme Specificity 5’→3’ FastDigest® enzyme Page Conventional enzyme PageCommercially available isos-chizomers from other vendors
BglII A↓GATCT FastDigest® BglII 21 BglII 90BisI GC↓NGC [BlsI], GluIBlnI C↓CTAGG FastDigest® AvrII (XmaJI) 18 XmaJI (AvrII) 156 AspA2I, AvrIIBlpI GC↓TNAGC FastDigest® BlpI (Bpu1102I) 21 Bpu1102I (BlpI) 93 BlpI, Bsp1720I, CelIIBlsI GCN↓GC [BisI], [GluI]BmcAI AGT↓ACT FastDigest® ScaI 61 ScaI 144 AssI, ZrmI
Bme1390I CC↓NGG FastDigest® ScrFI (Bme1390I) 61 Bme1390I (ScrFI) 91BmrFI, [BssKI], [BstSCI], MspR9I, ScrFI, [StyD4I]
Bme1580I GKGCM↓C FastDigest® Bme1580I (BseSI) 21 BseSI (Bme1580I) 96 BaeGI, BstSLIBme18I G↓GWCC FastDigest® AvaII (Eco47I) 18 Eco47I (AvaII) 111 AvaII, SinI, VpaK11BIBmeRI GACNNN↓NNGTC FastDigest® Eam1105I 35 Eam1105I (AhdI) 109 AhdI, AspEI, DriI, EclHKIBmeT110I CY↓CGRG [FastDigest® AvaI (Eco88I)m](C↓YCGRG) 17 [Eco88I (AvaI)m](C↓YCGRG) 113 [Ama87I], [AvaIm], [BsiHKCI], [BsoBI]BmgBI CACGTC(-3/-3)↓ AjiI (BmgBI) 82 BtrIBmgT120I GG↓NCC [FastDigest® Sau96I (Cfr13I)](G↓GNCC) 60 [Cfr13I (Sau96I)](G↓GNCC) 106 [AspS9I], [PspPI], [Sau96I]BmiI GGN↓NCC FastDigest® NlaIV (BspLI) 51 BspLI (NlaIV) 100 NlaIV, PspN4IBmrFI CC↓NGG FastDigest® ScrFI (Bme1390I) 61 Bme1390I (ScrFI) 91 [BssKI], [BstSCI], MspR9I, ScrFI, [StyD4I]BmrI ACTGGG(5/4)↓ BfiI (BmrI) 89 BmuI
BmtI GCTAG↓CFastDigest® BmtI (BspOI), [FastDigest® NheIm](G↓CTAGC)
2250
BspOI (BmtI), [NheIm](G↓CTAGC)
100132
[AsuNHI], BmtI
BmuI ACTGGG(5/4)↓ BfiI (BmrI) 89 BmrIBoxI GACNN↓NNGTC FastDigest® PshAI (BoxI) 55 BoxI (PshAI) 91 BstPAI, PshAIBpiI GAAGAC(2/6)↓ FastDigest® BbsI (BpiI) 19 BpiI (BbsI) 92 BbsI, BpuAI, BstV2IBplI ↓(8/13)GAG(N)5CTC(13/8)↓ FastDigest® BplI 22 BplI 92BpmI CTGGAG(16/14)↓ FastDigest® BpmI (GsuI)m 22 GsuI (BpmI)m 119 BpmIBpu10I CCTNAGC(-5/-2)↓ FastDigest® Bpu10I 23 Bpu10I 93Bpu1102I GC↓TNAGC FastDigest® BlpI (Bpu1102I) 21 Bpu1102I (BlpI) 93 BlpI, Bsp1720I, CelIIBpu14I TT↓CGAA FastDigest® Bsp119I 27 Bsp119I (BstBI) 99 AsuII, BspT104I, BstBI, Csp45I, NspV, SfuIBpuAI GAAGAC(2/6)↓ FastDigest® BbsI (BpiI) 19 BpiI (BbsI) 92 BbsI, BstV2IBpuEI CTTGAG(16/14)↓BpuMI CC↓SGG FastDigest® NciI (BcnI) 49 BcnI (NciI) 88 AsuC2I, NciIBpvUI CGAT↓CG FastDigest® PvuI 57 PvuI 141 MvrI, Ple19I
Bsa29I AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103BanIII, BseCI, BshVI, BspDI, BspXI, BsuTUI, ClaI
BsaAI YAC↓GTR FastDigest® BsaAI (Ppu21I) 23 Ppu21I (BsaAI) 138 BsaAI, BstBAIBsaBI GATNN↓NNATC FastDigest® BsaBI (BseJI) 23 BseJI (BsaBI) 94 BsaBI, Bse8IBsaHI GR↓CGYC FastDigest® BsaHI (Hin1I)m 24 Hin1I (BsaHI)m 120 AcyI, BsaHI, BssNI, BstACI, Hsp92IBsaI GGTCTC(1/5)↓ FastDigest® Eco31I 36 Eco31I (BsaI) 110 Bso31I, BspTNIBsaJI C↓CNNGG FastDigest® BsaJI (BseDI) 24 BseDI (BsaJI) 94 BsaJI, BssECIBsaMI GAATGC(1/-1)↓ FastDigest® Mva1269I 49 Mva1269I (BsmI) 131 BsmI, PctIBsaWI W↓CCGGWBsaXI ↓(9/12)AC(N)5CTCC(10/7)↓Bsc4I CCNNNNN↓NNGG FastDigest® BslI (BseLI) 26 BseLI (BslI) 95 AfiI, BslIBse118I R↓CCGGY FastDigest® BsrFI (Cfr10I) 30 Cfr10I (BsrFI) 105 BsrFI, BssAIBse1I ACTGG(1/-1)↓ FastDigest® BseNI 25 BseNI (BsrI) 96 BsrI, BsrSIBse21I CC↓TNAGG FastDigest® Bsu36I (Eco81I) 32 Eco81I (Bsu36I) 113 AxyI, Bsu36IBse3DI GCAATG(2/0)↓ FastDigest® BsrDI (BseMI) 30 BseMI (BsrDI) 95 BsrDIBse8I GATNN↓NNATC FastDigest® BsaBI (BseJI) 23 BseJI (BsaBI) 94 BsaBIBseAI T↓CCGGA FastDigest® Kpn2Im 44 Kpn2I (BspEI)m 124 AccIIIm, Aor13HI, Bsp13I, BspEIm, MroIm
BseBI CC↓WGG FastDigest® MvaI 48[EcoRII](↓CCWGG), MvaI (BstNI)
116130
[AjnI], Bst2UI, BstNI, BstOI, [Psp6I], [PspGI]
BseCI AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103BanIII, Bsa29I, BshVI, BspDI, BspXI, BsuTUI, ClaI
BseDI C↓CNNGG FastDigest® BsaJI (BseDI) 24 BseDI (BsaJI) 94 BsaJI, BssECI
BseGI GGATG(2/0)↓ FastDigest® BseGI, [FastDigest® FokI](GGATG(9/13)↓)
2438
BseGI (BtsCI) 94 BstF5I, BtsCI, [FokI]
BseJI GATNN↓NNATC FastDigest® BsaBI (BseJI) 23 BseJI (BsaBI) 94 BsaBI, Bse8IBseLI CCNNNNN↓NNGG FastDigest® BslI (BseLI) 26 BseLI (BslI) 95 AfiI, Bsc4I, BslIBseMI GCAATG(2/0)↓ FastDigest® BsrDI (BseMI) 30 BseMI (BsrDI) 95 Bse3DI, BsrDIBseMII CTCAG(10/8)↓ FastDigest® BspCNI (BseMII) 29 BseMII (BspCNI) 95 [BspCNI]BseNI ACTGG(1/-1)↓ FastDigest® BseNI 25 BseNI (BsrI) 96 Bse1I, BsrI, BsrSIBsePI G↓CGCGC FastDigest® BssHII (PteI) 31 PauI (BssHII) 135 BssHIIBseRI GAGGAG(10/8)↓BseSI GKGCM↓C FastDigest® Bme1580I (BseSI) 21 BseSI (Bme1580I) 96 BaeGI, BstSLI
BseXI GCAGC(8/12)↓ FastDigest® BseXI, FastDigest® BbvI (Lsp1109I)
2519
BseXI (BbvI), Lsp1109I (BbvI)
96125
BbvI, BstV1I
BseX3I C↓GGCCG FastDigest® EagI (Eco52I) 34 Eco52I (EagI) 111 BstZI, EagI, EclXIBseYI CCCAGC(-5/-1)↓ [FastDigest® PspFI] (CCCAGC(-1/-5)↓) 56 [GsaI]
210
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.25. Commercially available restriction enzymes.
(continued on next page)
Enzyme Specificity 5’→3’ FastDigest® enzyme Page Conventional enzyme PageCommercially available isos-chizomers from other vendors
BsgI GTGCAG(16/14)↓Bsh1236I CG↓CG FastDigest® Bsh1236I 25 Bsh1236I (BstUI) 97 AccII, BspFNI, BstFNI, BstUI, MvnIBsh1285I CGRY↓CG FastDigest® BsiEI (Bsh1285I) 26 Bsh1285I (BsiEI) 97 BsiEIm, BstMCIBshFI GG↓CC FastDigest® HaeIII (BsuRI) 40 BsuRI (HaeIII) 103 BsnI, BspANI, HaeIII, PhoIBshNI G↓GYRCC FastDigest® BanI (BshNI) 19 BshNI (BanI) 97 AccB1I, BanI, BspT107IBshTI A↓CCGGT FastDigest® AgeI (BshTI) 13 BshTI (AgeI) 98 AgeI, AsiGI, CspAIm, PinAI
BshVI AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103BanIII, Bsa29I, BseCI, BspDI, BspXI, BsuTUI, ClaI
BsiEI CGRY↓CG FastDigest® BsiEI (Bsh1285I)m 26 Bsh1285I (BsiEI)m 97 BsiEI, BstMCIBsiHKAI GWGCW↓C FastDigest® Alw21I 15 Alw21I (BsiHKAI) 84 Bbv12IBsiHKCI C↓YCGRG FastDigest® AvaI (Eco88I) 17 Eco88I (AvaI) 113 Ama87I, AvaI, [BmeT110I], BsoBI
BsiSI C↓CGGFastDigest® HpaIIm, FastDigest® MspI
4248
HpaIIm, MspI (HpaII)
122129
HapIIm
BsiWI C↓GTACG FastDigest® BsiWI (Pfl23II) 26 Pfl23II (BsiWI) 137 BsiWI, PspLIBslFI GGGAC(10/14)↓ FastDigest® BsmFI (FaqI) 27 FaqI (BsmFI) 118 BsmFIBslI CCNNNNN↓NNGG FastDigest® BslI (BseLI) 26 BseLI (BslI) 95 AfiI, Bsc4I, BslIBsmAI GTCTC(1/5)↓ FastDigest® Alw26Im 15 Alw26I (BsmAI)m 85 BstMAIBsmBI CGTCTC(1/5)↓ FastDigest® BsmBI (Esp3I) 27 Esp3I (BsmBI) 117 BsmBIBsmFI GGGAC(10/14)↓ FastDigest® BsmFI (FaqI) 27 FaqI (BsmFI) 118 BslFI, BsmFIBsmI GAATGC(1/-1)↓ FastDigest® Mva1269I 49 Mva1269I (BsmI) 131 BsaMI, PctIBsnI GG↓CC FastDigest® HaeIII (BsuRI) 40 BsuRI (HaeIII) 103 BshFI, BspANI, HaeIII, PhoIBso31I GGTCTC(1/5)↓ FastDigest® Eco31I 36 Eco31I (BsaI) 110 BsaI, BspTNIBsoBI C↓YCGRG FastDigest® AvaI (Eco88I)m 17 Eco88I (AvaI)m 113 Ama87I, AvaIm, [BmeT110I], BsiHKCI
Bsp119I TT↓CGAA FastDigest® Bsp119I 27 Bsp119I (BstBI) 99AsuIIm, Bpu14I, BspT104I, BstBI, Csp45I, NspV, SfuIm
Bsp120I G↓GGCCC[FastDigest® ApaI](GGGCC↓C), FastDigest® Bsp120I
1628
[ApaI](GGGCC↓C), Bsp120I (PspOMI)
8599
PspOMI
Bsp1286I GDGCH↓C FastDigest® Bsp1286I (SduI) 28 SduI (Bsp1286I) 145 Bsp1286I, MhlIBsp1407I T↓GTACA FastDigest® Bsp1407I 28 Bsp1407I (BsrGI) 100 BsrGI, BstAUI
Bsp143I ↓GATCFastDigest® Sau3AI (Bsp143I), FastDigest® MboIm
6044
Bsp143I (Sau3AI), MboIm
99127
BfuCI, BssMI, [BstKTIm], BstMBI, DpnIIm, Kzo9I, NdeIIm, Sau3AIm
Bsp13I T↓CCGGA FastDigest® Kpn2I 44 Kpn2I (BspEI) 124 AccIII, Aor13HI, BseAI, BspEI, MroIBsp1720I GC↓TNAGC FastDigest® BlpI (Bpu1102I) 21 Bpu1102I (BlpI) 93 BlpI, CelIIBsp19I C↓CATGG FastDigest® NcoI 50 NcoI 131Bsp68I TCG↓CGA FastDigest® NruI (RruI) 52 Bsp68I (NruI) 98 BtuMI, NruIBspACI CCGC(-3/-1)↓ FastDigest® AciI (SsiI) 12 SsiI (AciI) 149 AciIBspANI GG↓CC FastDigest® HaeIII (BsuRI) 40 BsuRI (HaeIII) 103 BshFI, BsnI, HaeIII, PhoI
BspCNI CTCAG(9/7)↓ [FastDigest® BspCNI (BseMII)](CTCAG(10/8)↓)
29 [BseMII (BspCNI)](CTCAG(10/8)↓) 95 BspCNI
BspDI AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103BanIII, Bsa29I, BseCI, BshVI, BspXI, BsuTUI, ClaI
BspEI T↓CCGGA FastDigest® Kpn2Im 44 Kpn2I (BspEI)m 124 AccIIIm, Aor13HI, BseAIm, Bsp13I, MroIm
BspFNI CG↓CG FastDigest® Bsh1236I 25 Bsh1236I (BstUI) 97 AccII, BstFNI, BstUI, MvnIBspHI T↓CATGA FastDigest® BspHI (PagI)m 29 PagI (BspHI)m 135 BspHI, CciI, RcaIBspLI GGN↓NCC FastDigest® NlaIV (BspLI) 51 BspLI (NlaIV) 95 BmiI, NlaIVm, PspN4IBspMAI CTGCA↓G FastDigest® PstI 56 PstI 140BspMI ACCTGC(4/8)↓ FastDigest® BspMI (BveI) 29 BveI (BspMI) 104 Acc36I, BfuAI, BspMI
BspOI GCTAG↓CFastDigest® BmtI (BspOI), [FastDigest® NheI](G↓CTAGC)
2250
BspOI (BmtI), [NheI](G↓CTAGC)
100132
[AsuNHI], BmtI
BspPI GGATC(4/5)↓ BspPI (AlwI) 101 AclWI, AlwIBspQI GCTCTTC(1/4)↓ FastDigest® SapI (LguI) 59 LguI (SapI) 125 PciSI, SapIBspT104I TT↓CGAA FastDigest® Bsp119I 27 Bsp119I (BstBI) 99 AsuIIm, Bpu14I, BstBI, Csp45I, NspV, SfuIm
BspT107I G↓GYRCC FastDigest® BanI (BshNI) 19 BshNI (BanI) 97 AccB1I, BanIBspTI C↓TTAAG FastDigest® AflII (BspTI) 13 BspTI (AflII) 101 AflII, BfrI, Bst98I, BstAFI, MspCI, Vha464IBspTNI GGTCTC(1/5)↓ FastDigest® Eco31I 36 Eco31I (BsaI) 110 BsaI, Bso31I
BspXI AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103BanIII, Bsa29I, BseCI, BshVI, BspDI, BsuTUI, ClaI
BsrBI CCGCTC(-3/-3)↓ FastDigest® BsrBI (MbiI)m 30 MbiI (BsrBI)m 126 AccBSI, BsrBIBsrDI GCAATG(2/0)↓ FastDigest® BsrDI (BseMI) 30 BseMI (BsrDI) 95 Bse3DI, BsrDIBsrFI R↓CCGGY FastDigest® BsrFI (Cfr10I) 30 Cfr10I (BsrFI) 105 Bse118I, BsrFI, BssAIm
BsrGI T↓GTACA FastDigest® Bsp1407I 28 Bsp1407I (BsrGI) 100 BstAUIBsrI ACTGG(1/-1)↓ FastDigest® BseNI 25 BseNI (BsrI) 96 Bse1I, BsrSIBsrSI ACTGG(1/-1)↓ FastDigest® BseNI 25 BseNI (BsrI) 96 Bse1I, BsrIBssAI R↓CCGGY FastDigest® BsrFI (Cfr10I)m 30 Cfr10I (BsrFI)m 105 Bse118I, BsrFIm
BssECI C↓CNNGG FastDigest® BsaJI (BseDI) 24 BseDI (BsaJI) 94 BsaJIBssHII G↓CGCGC FastDigest® BssHII (PteI) 31 PauI (BssHII) 135 BsePI, BssHIIBssKI ↓CCNGG [FastDigest® ScrFI (Bme1390I)](CC↓NGG) 61 [Bme1390I (ScrFI)](CC↓NGG) 91 [BmrFI], BstSCI, [MspR9I], [ScrFI], StyD4I
211
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.25. Commercially available restriction enzymes.
(continued on next page)
Enzyme Specificity 5’→3’ FastDigest® enzyme Page Conventional enzyme PageCommercially available isos-chizomers from other vendors
BssMI ↓GATCFastDigest® Sau3AI (Bsp143I), FastDigest® MboI
6044
Bsp143I (Sau3AI), MboI
99127
BfuCI, [BstKTI], BstMBI, DpnII, Kzo9I, NdeII, Sau3AI
BssNAI GTA↓TAC FastDigest® BstZ17I (Bst1107I) 31 Bst1107I (BstZ17I) 102 BstZ17IBssNI GR↓CGYC FastDigest® BsaHI (Hin1I) 24 Hin1I (BsaHI) 120 AcyI, BsaHI, BstACI, Hsp92IBssSI CACGAG(-5/-1)↓ BauI (BssSI) 87 Bst2BIBssT1I C↓CWWGG FastDigest® StyI (Eco130I) 65 Eco130I (StyI) 114 EcoT14I, ErhI, StyIBst1107I GTA↓TAC FastDigest® BstZ17I (Bst1107I) 31 Bst1107I (BstZ17I) 102 BssNAI, BstZ17IBst2BI CACGAG(-5/-1)↓ BauI (BssSI) 87 BssSI
Bst2UI CC↓WGG FastDigest® MvaI 48[EcoRIIm](↓CCWGG), MvaI (BstNI)
116130
[AjnI], BseBI, BstNI, BstOI, [Psp6I], [PspGIm]
Bst4CI ACN↓GT FastDigest® TaaI 65 TaaI (HpyCH4III) 150 HpyCH4IIIBst6I CTCTTC(1/4)↓ FastDigest® EarI (Eam1104I) 35 Eam1104I (EarI) 109 EarIBst98I C↓TTAAG FastDigest® AflII (BspTI) 13 BspTI (AflII) 101 AflII, BfrI, BstAFI, MspCI, Vha464IBstACI GR↓CGYC FastDigest® BsaHI (Hin1I) 24 Hin1I (BsaHI) 120 AcyI, BsaHI, BssNI, Hsp92IBstAFI C↓TTAAG FastDigest® AflII (BspTI) 13 BspTI (AflII) 101 AflII, BfrI, Bst98I, MspCI, Vha464IBstAPI GCANNNN↓NTGCBstAUI T↓GTACA FastDigest® Bsp1407I 28 Bsp1407I (BsrGI) 100 BsrGIBstBAI YAC↓GTR FastDigest® BsaAI (Ppu21I) 23 Ppu21I (BsaAI) 138 BsaAI
BstBI TT↓CGAA FastDigest® Bsp119I 27 Bsp119I (BstBI) 99AsuIIm, Bpu14I, BspT104I, Csp45I, NspVm, SfuIm
BstC8I GCN↓NGC Cac8IBstDEI C↓TNAG FastDigest® DdeI (HpyF3I) 33 HpyF3I (DdeI) 123 DdeIBstDSI C↓CRYGG BtgIBstEII G↓GTNACC FastDigest® Eco91I 36 Eco91I (BstEII) 114 BstPI, EcoO65I, PspEIBstENI CCTNN↓NNNAGG FastDigest® EcoNI (XagI) 36 XagI (EcoNI) 155 EcoNI
BstF5I GGATG(2/0)↓ FastDigest® BseGI, [FastDigest® FokI](GGATG(9/13)↓)
2438
BseGI (BtsCI) 94 BtsCI, [FokI]
BstFNI CG↓CG FastDigest® Bsh1236I 25 Bsh1236I (BstUI) 97 AccII, BspFNI, BstUI, MvnIBstH2I RGCGC↓Y FastDigest® HaeII (BfoI) 41 HaeII
BstHHI GCG↓CFastDigest® HhaI, [FastDigest® HinP1I (Hin6I)](G↓CGC)
4042
HhaI, [Hin6I (HinP1I)](G↓CGC)
120121
AspLEI, CfoI, [HinP1I], [HspAI]
BstKTI GAT↓C[FastDigest® Sau3AI (Bsp143I)m](↓GATC), [FastDigest® MboI](↓GATC)
6044
[Bsp143I (Sau3AI)m](↓GATC), [MboI](↓GATC)
99127
[BfuCIm], [BssMI], [BstMBI], [DpnII], [Kzo9Im], [NdeIIm], [Sau3AIm]
BstMAI GTCTC(1/5)↓ FastDigest® Alw26I 15 Alw26I (BsmAI) 85 BsmAI
BstMBI ↓GATCFastDigest® Sau3AI (Bsp143I), FastDigest® MboI
6044
Bsp143I (Sau3AI), MboI
99127
BfuCI, BssMI, [BstKTI], DpnII, Kzo9I, NdeII, Sau3AI
BstMCI CGRY↓CG FastDigest® BsiEI (Bsh1285I) 26 Bsh1285I (BsiEI) 97 BsiEIBstMWI GCNNNNN↓NNGC FastDigest® HpyF10VI 43 HpyF10VI (MwoI) 124 MwoI
BstNI CC↓WGG FastDigest® MvaI 48[EcoRIIm](↓CCWGG), MvaI (BstNI)
116130
[AjnI], BseBI, Bst2UI, BstOI, [Psp6I], [PspGIm]
BstNSI RCATG↓Y FastDigest® NspI (XceI) 53 XceI (NspI) 156 NspI
BstOI CC↓WGG FastDigest® MvaI 48[EcoRIIm](↓CCWGG), MvaI (BstNI)
116130
[AjnI], BseBI, Bst2UI, BstNI, [Psp6I], [PspGIm]
BstPAI GACNN↓NNGTC FastDigest® PshAI (BoxI) 55 BoxI (PshAI) 91 PshAIBstPI G↓GTNACC FastDigest® Eco91I 36 Eco91I (BstEII) 114 BstEII, EcoO65I, PspEIBstSCI ↓CCNGG [FastDigest® ScrFI (Bme1390I)](CC↓NGG) 61 [Bme1390I (ScrFI)](CC↓NGG) 91 [BmrFI], BssKI, [MspR9I], [ScrFI], StyD4IBstSFI C↓TRYAG FastDigest® SfcI (BfmI) 62 BfmI (SfcI) 89 SfcIBstSLI GKGCM↓C FastDigest® Bme1580I (BseSI) 21 BseSI (Bme1580I) 96 BaeGIBstSNI TAC↓GTA FastDigest® SnaBI (Eco105I) 63 Eco105I (SnaBI) 114 SnaBIBstUI CG↓CG FastDigest® Bsh1236I 25 Bsh1236I (BstUI) 97 AccII, BspFNI, BstFNI, MvnI
BstV1I GCAGC(8/12)↓ FastDigest® BbvI (Lsp1109I), FastDigest® BseXI
1925
Lsp1109I (BbvI), BseXI (BbvI)
12596
BbvI
BstV2I GAAGAC(2/6)↓ FastDigest® BbsI (BpiI) 19 BpiI (BbsI) 92 BbsI, BpuAIBstX2I R↓GATCY FastDigest® PsuI 56 PsuI (BstYI) 140 BstYI, MflI, XhoIIBstXI CCANNNNN↓NTGG FastDigest® BstXI 31 BstXI 102BstYI R↓GATCY FastDigest® PsuI 56 PsuI (BstYI) 140 BstX2I, MflIm, XhoIIBstZ17I GTA↓TAC FastDigest® BstZ17I (Bst1107I) 31 Bst1107I (BstZ17I) 102 BssNAI, BstZ17IBstZI C↓GGCCG FastDigest® EagI (Eco52I) 34 Eco52I (EagI) 111 BseX3I, EagI, EclXI
Bsu15I AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103BanIII, Bsa29I, BseCI, BshVI, BspDI, BspXI, BsuTUI, ClaI
Bsu36I CC↓TNAGG FastDigest® Bsu36I (Eco81I) 32 Eco81I (Bsu36I) 113 AxyI, Bse21I, Bsu36IBsuRI GG↓CC FastDigest® HaeIII (BsuRI) 40 BsuRI (HaeIII) 103 BshFI, BsnI, BspANI, HaeIII, PhoI
BsuTUI AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103BanIII, Bsa29I, BseCI, BshVI, BspDI, BspXI, ClaI
BtgI C↓CRYGG BstDSIBtgZI GCGATG(10/14)↓BtrI CACGTC(-3/-3)↓ AjiI (BmgBI) 82 BmgBI
212
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.25. Commercially available restriction enzymes.
(continued on next page)
Enzyme Specificity 5’→3’ FastDigest® enzyme Page Conventional enzyme PageCommercially available isos-chizomers from other vendors
BtsCI GGATG(2/0)↓ FastDigest® BseGI, [FastDigest® FokI](GGATG(9/13)↓)
2438
BseGI (BtsCI) 94 BstF5I, [FokI]
BtsI GCAGTG(2/0)↓BtuMI TCG↓CGA FastDigest® NruI (RruI) 52 Bsp68I (NruI) 98 NruIBveI ACCTGC(4/8)↓ FastDigest® BspMI (BveI) 29 BveI (BspMI) 104 Acc36I, BfuAI, BspMICac8I GCN↓NGC BstC8ICaiI CAGNNN↓CTG FastDigest® AlwNI (CaiI) 15 CaiI (AlwNI) 104 AlwNICciI T↓CATGA FastDigest® BspHI (PagI) 29 PagI (BspHI) 135 BspHI, RcaICciNI GC↓GGCCGC FastDigest® NotI 52 NotI 133CelII GC↓TNAGC FastDigest® BlpI (Bpu1102I) 21 Bpu1102I (BlpI) 93 BlpI, Bsp1720I
CfoI GCG↓CFastDigest® HhaI, [FastDigest® HinP1I (Hin6I)](G↓CGC)
4042
HhaI, [Hin6I (HinP1I)](G↓CGC)
120121
AspLEI, BstHHI, [HinP1I], [HspAI]
Cfr10I R↓CCGGY FastDigest® BsrFI (Cfr10I) 30 Cfr10I (BsrFI) 105 Bse118I, BsrFI, BssAIm
Cfr13I G↓GNCC FastDigest® Sau96I (Cfr13I) 60 Cfr13I (Sau96I) 106 AspS9I, [BmgT120I], PspPI, Sau96ICfr42I CCGC↓GG Cfr42I (SacII) 106 KspI, SacII, Sfr303I, SgrBI, SstII
Cfr9I C↓CCGGG [FastDigest® SmaIm](CCC↓GGG) 63Cfr9I (XmaI), [SmaIm](CCC↓GGG)
105147
TspMIm, XmaCI, XmaIm
CfrI Y↓GGCCR CfrI (EaeI) 105 AcoI, EaeI
ClaI AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103BanIII, Bsa29I, BseCI, BshVI, BspDI, BspXI, BsuTUI, ClaI
CpoI CG↓GWCCG FastDigest® RsrII (CpoI) 58 CpoI (RsrII) 106 CspI, Rsr2I, RsrIICseI GACGC(5/10)↓ FastDigest® HgaI (CseI) 40 CseI (HgaI) 107 HgaI
Csp45I TT↓CGAA FastDigest® Bsp119I 27 Bsp119I (BstBI) 99AsuII, Bpu14I, BspT104I, BstBI, NspVm, SfuI
Csp6I G↓TACFastDigest® Csp6I, [FastDigest® RsaIm](GT↓AC)
3258
Csp6I (CviQI), [RsaIm](GT↓AC)
107141
[AfaIm], CviQI, RsaNI
CspAI A↓CCGGT FastDigest® AgeI (BshTI)m 13 BshTI (AgeI)m 98 AgeIm, AsiGI, PinAICspCI ↓(11/13)CAA(N)5GTGG(12/10)↓CspI CG↓GWCCG FastDigest® RsrII (CpoI) 58 CpoI (RsrII) 106 Rsr2I, RsrIICviAII C↓ATG [FastDigest® NlaIII (Hin1II)](CATG↓) 51 [Hin1II (NlaIII)](CATG↓) 120 [FaeI], [FatIm][Hsp92II], [NlaIII]CviJI RG↓CY CviKI-1CviKI-1 RG↓CY CviJI
CviQI G↓TACFastDigest® Csp6I, [FastDigest® RsaIm](GT↓AC)
3258
Csp6I (CviQI), [RsaIm](GT↓AC)
107141
[AfaIm], RsaNI
DdeI C↓TNAG FastDigest® DdeI (HpyF3I) 33 HpyF3I (DdeI) 123 BstDEI, DdeI
DinI GGC↓GCC FastDigest® EheI 117EheI (SfoI), [SspDI (KasI)](G↓GCGCC)
117150
[BbeI], EgeI, [KasI], [Mly113I], [NarI], SfoI
DpnI Gm6A↓TC FastDigest® DpnI 33 DpnI 108 MalI
DpnII ↓GATCFastDigest® Sau3AI (Bsp143I)m, FastDigest® MboIm
6044
[Bsp143I (Sau3AI)m], MboIm
99127
BfuCIm, BssMI, [BstKTIm], BstMBI, Kzo9Im, NdeII, Sau3AIm
DraI TTT↓AAA FastDigest® DraI 33 DraI 108DraII RG↓GNCCY FastDigest® EcoO109I 37 EcoO109I (DraII) 115DraIII CACNNN↓GTG FastDigest® DraIII (AdeI) 34 AdeI (DraIII) 82 DraIIIDrdI GACNNNN↓NNGTC FastDigest® DrdI (AasI) 34 AasI (DrdI) 80 DrdI, DseDIDriI GACNNN↓NNGTC FastDigest® Eam1105I 35 Eam1105I (AhdI) 109 AhdI, AspEI, BmeRI, EclHKIDseDI GACNNNN↓NNGTC FastDigest® DrdI (AasI) 34 AasI (DrdI) 80 DrdIEaeI Y↓GGCCR CfrI (EaeI) 105 AcoIEagI C↓GGCCG FastDigest® EagI (Eco52I) 34 Eco52I (EagI) 111 BseX3I, BstZI, EagI, EclXIEam1104I CTCTTC(1/4)↓ FastDigest® EarI (Eam1104I) 35 Eam1104I (EarI) 109 Bst6I, EarIEam1105I GACNNN↓NNGTC FastDigest® Eam1105I 35 Eam1105I (AhdI) 109 AhdI, AspEI, BmeRI, DriI, EclHKIEarI CTCTTC(1/4)↓ FastDigest® EarI (Eam1104I) 35 Eam1104I (EarI) 109 Bst6I, EarIEciI GGCGGA(11/9)↓
Ecl136II GAG↓CTCFastDigest® Ecl136II, [FastDigest® SacIm](GAGCT↓C)
3558
Ecl136II (EcoICRI), [SacIm](GAGCT↓C)
109142
Eco53kIm, EcoICRI, [Psp124BI], [SstIm]
EclHKI GACNNN↓NNGTC FastDigest® Eam1105I 35 Eam1105I (AhdI) 109 AhdI, AspEI, BmeRI, DriIEclXI C↓GGCCG FastDigest® EagI (Eco52I) 34 Eco52I (EagI) 111 BseX3I, BstZI, EagIEco105I TAC↓GTA FastDigest® SnaBI (Eco105I) 63 Eco105I (SnaBI) 114 BstSNI, SnaBIEco130I C↓CWWGG FastDigest® StyI (Eco130I) 65 Eco130I (StyI) 114 BssT1I, EcoT14I, ErhI, StyIEco147I AGG↓CCT FastDigest® StuI (Eco147I) 64 Eco147I (StuI) 115 AatI, PceI, SseBI, StuIEco24I GRGCY↓C Eco24I (BanII) 110 BanII, EcoT38I, FriOIEco31I GGTCTC(1/5)↓ FastDigest® Eco31I 36 Eco31I (BsaI) 110 BsaI, Bso31I, BspTNIEco32I GAT↓ATC FastDigest® EcoRV (Eco32I) 37 Eco32I (EcoRV) 110 EcoRVEco47I G↓GWCC FastDigest® AvaII (Eco47I) 18 Eco47I (AvaII) 111 AvaII, Bme18I, SinI, VpaK11BIEco47III AGC↓GCT FastDigest® AfeI (Eco47III) 13 Eco47III (AfeI) 111 AfeI, Aor51HIEco52I C↓GGCCG FastDigest® EagI (Eco52I) 34 Eco52I (EagI) 111 BseX3I, BstZI, EagI, EclXI
Eco53kI GAG↓CTCFastDigest® Ecl136IIm, [FastDigest® SacIm](GAGCT↓C)
3558
Ecl136II (EcoICRI)m, [SacIm](GAGCT↓C)
109142
EcoICRI, [Psp124BI], [SstI]
Eco57I CTGAAG(16/14)↓ FastDigest® AcuI (Eco57I) 12 Eco57I (AcuI) 112 AcuI
213
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.25. Commercially available restriction enzymes.
(continued on next page)
Enzyme Specificity 5’→3’ FastDigest® enzyme Page Conventional enzyme PageCommercially available isos-chizomers from other vendors
Eco57MI CTGRAG(16/14)↓ Eco57MI 112Eco72I CAC↓GTG FastDigest® PmlI (Eco72I) 54 Eco72I (PmlI) 113 AcvI, BbrPI, PmaCI, PmlI, PspCIEco81I CC↓TNAGG FastDigest® Bsu36I (Eco81I) 32 Eco81I (Bsu36I) 113 AxyI, Bse21I, Bsu36I
Eco88I C↓YCGRG FastDigest® AvaI (Eco88I) 17 Eco88I (AvaI) 113Ama87I, AvaIm, [BmeT110Im], BsiHKCI, BsoBIm
Eco91I G↓GTNACC FastDigest® Eco91I 36 Eco91I (BstEII) 114 BstEII, BstPI, EcoO65I, PspEI
EcoICRI GAG↓CTCFastDigest® Ecl136II, [FastDigest® SacI](GAGCT↓C)
3558
Ecl136II (EcoICRI), [SacI](GAGCT↓C)
109142
Eco53kI, [Psp124BI], [SstI]
EcoNI CCTNN↓NNNAGG FastDigest® EcoNI (XagI) 36 XagI (EcoNI) 155 BstENI, EcoNIEcoO109I RG↓GNCCY FastDigest® EcoO109I 37 EcoO109I (DraII) 115 DraIIEcoO65I G↓GTNACC FastDigest® Eco91I 36 Eco91I (BstEII) 114 BstEII, BstPI, PspEIEcoP15I CAGCAG(25/27)↓EcoRI G↓AATTC FastDigest® EcoRI 37 EcoRI 116
EcoRII ↓CCWGG [FastDigest® MvaIm](CC↓WGG) 48EcoRII, [MvaI (BstNI)m](CC↓WGG)
116130
AjnIm, [BseBI], [Bst2UIm], [BstNIm], [BstOIm], Psp6I, PspGI
EcoRV GAT↓ATC FastDigest® EcoRV (Eco32I) 37 Eco32I (EcoRV) 110 EcoRVEcoT14I C↓CWWGG FastDigest® StyI (Eco130I) 65 Eco130I (StyI) 114 BssT1I, ErhI, StyIEcoT22I ATGCA↓T FastDigest® NsiI (Mph1103I) 52 Mph1103I (NsiI) 129 NsiIm, Zsp2IEcoT38I GRGCY↓C Eco24I (BanII) 110 BanII, FriOI
EgeI GGC↓GCC FastDigest® EheI 38EheI (SfoI), [SspDI (KasI)](G↓GCGCC)
117150
[BbeI], DinI, [KasI], [Mly113I], [NarI], SfoI
EheI GGC↓GCC FastDigest® EheI 38EheI (SfoI), [SspDI (KasI)](G↓GCGCC)
117150
[BbeI], DinI, EgeI, [KasI], [Mly113I], [NarI], SfoIm
ErhI C↓CWWGG FastDigest® StyI (Eco130I) 65 Eco130I (StyI) 114 BssT1I, EcoT14I, StyIEsp3I CGTCTC(1/5)↓ FastDigest® BsmBI (Esp3I) 27 Esp3I (BsmBI) 117 BsmBIFaeI CATG↓ FastDigest® NlaIII (Hin1II) 51 Hin1II (NlaIII) 120 [CviAII], [FatI], Hsp92II, NlaIIIFaiI YA↓TRFaqI GGGAC(10/14)↓ FastDigest® BsmFI (FaqI) 27 FaqI (BsmFI) 118 BslFI, BsmFIFalI ↓(8/13)AAG(N)5CTT(13/8)↓FatI ↓CATG [FastDigest® NlaIII (Hin1II)](CATG↓) 51 [Hin1II (NlaIII)](CATG↓) 120 [CviAIIm], [FaeI], [Hsp92II], [NlaIIIm]FauI CCCGC(4/6)↓ SmuI (FauI) 148FauNDI CA↓TATG FastDigest® NdeI 50 NdeI 132FbaI T↓GATCA FastDigest® BclI 20 BclI 87 Ksp22IFblI GT↓MKAC FastDigest® AccI (XmiI) 11 XmiI (AccI) 157 AccIFnu4HI GC↓NGC FastDigest® Fnu4HI (SatI) 38 SatI (Fnu4HI) 143 Fnu4HI, Fsp4HI, ItaIm
FokI GGATG(9/13)↓ [FastDigest® BseGI](GGATG(2/0)↓), FastDigest® FokI
2438
[BseGI (BtsCI)](GGATG(2/0)↓) 94 [BstF5I], [BtsCI], FokI
FriOI GRGCY↓C Eco24I (BanII) 110 BanII, EcoT38IFseI GGCCGG↓CC RigIFsp4HI GC↓NGC FastDigest® Fnu4HI (SatI) 38 SatI (Fnu4HI) 143 Fnu4HI, ItaIm
FspAI RTGC↓GCAY FastDigest® FspAI 39 FspAI 118FspBI C↓TAG FastDigest® BfaI (FspBI) 20 FspBI (BfaI) 119 BfaI, MaeI, XspIFspI TGC↓GCA FastDigest® FspI (NsbI) 39 NsbI (FspI) 133 Acc16I, AviII, FspIGlaI GC↓GCGluI GC↓NGC BisI, [BlsI]GsaI CCCAGC(-1/-5)↓ FastDigest® PspFI 56 [BseYI]GsuI CTGGAG(16/14)↓ FastDigest® BpmI (GsuI) 22 GsuI (BpmI) 119 BpmIm
HaeII RGCGC↓Y FastDigest® HaeII (BfoI) 39 BstH2I, HaeIIHaeIII GG↓CC FastDigest® HaeIII (BsuRI) 40 BsuRI (HaeIII) 103 BshFI, BsnI, BspANI, HaeIII, PhoI
HapII C↓CGGFastDigest® HpaII, FastDigest® MspIm
4248
HpaII, MspI (HpaII)m
122129
BsiSIm
HgaI GACGC(5/10)↓ FastDigest® HgaI (CseI) 40 CseI (HgaI) 107 HgaI
HhaI GCG↓CFastDigest® HhaI, [FastDigest® HinP1I (Hin6I)](G↓CGC)
4042
HhaI, [Hin6I (HinP1I)](G↓CGC)
120121
AspLEI, BstHHI, CfoI, [HinP1I], [HspAI]
Hin1I GR↓CGYC FastDigest® BsaHI (Hin1I) 24 Hin1I (BsaHI) 120 AcyI, BsaHIm, BssNI, BstACI, Hsp92IHin1II CATG↓ FastDigest® NlaIII (Hin1II) 51 Hin1II (NlaIII) 120 [CviAII], FaeI, [FatI], Hsp92II, NlaIIIHin4I ↓(8/13)GAY(N)5VTC(13/8)↓ Hin4I 121
Hin6I G↓CGC[FastDigest® HhaI](GCG↓C), FastDigest® HinP1I (Hin6I)
4042
[HhaI](GCG↓C), Hin6I (HinP1I)
120121
[AspLEI], [BstHHI], [CfoI], HinP1I, HspAI
HincII GTY↓RAC FastDigest® HincII 41 HincII (HindII) 121 HindIIHindII GTY↓RAC FastDigest® HincII 41 HincII (HindII) 121HindIII A↓AGCTT FastDigest® HindIII 41 HindIII 122HinfI G↓ANTC FastDigest® HinfI 41 HinfI 122
HinP1I G↓CGC[FastDigest® HhaI](GCG↓C), FastDigest® HinP1I (Hin6I)
4042
[HhaI](GCG↓C), Hin6I (HinP1I)
120121
[AspLEI], [BstHHI], [CfoI], HinP1I, HspAI
HpaI GTT↓AAC FastDigest® HpaI (KspAI)m 42 KspAI (HpaI)m 125 HpaI
HpaII C↓CGGFastDigest® HpaII, FastDigest® MspIm
4248
HpaII, MspI (HpaII)m
122129
BsiSIm, HapII
214
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.25. Commercially available restriction enzymes.
(continued on next page)
Enzyme Specificity 5’→3’ FastDigest® enzyme Page Conventional enzyme PageCommercially available isos-chizomers from other vendors
HphI GGTGA(8/7)↓ HphI 123 AsuHPIHpy166II GTN↓NAC FastDigest® Hpy8I 43 Hpy8I (MjaIV) 123Hpy188I TCN↓GAHpy188III TC↓NNGAHpy8I GTN↓NAC FastDigest® Hpy8I 43 Hpy8I (MjaIV) 123 Hpy166IIHpy99I CGWCG↓HpyAV CCTTC(6/5)↓HpyCH4III ACN↓GT FastDigest® TaaI 65 TaaI (HpyCH4III) 150 Bst4CIHpyCH4IV A↓CGT [FastDigest® TaiIm](ACGT↓) 66 [TaiI (MaeII)m](ACGT↓) 150 MaeIIHpyCH4V TG↓CAHpyF10VI GCNNNNN↓NNGC FastDigest® HpyF10VI 43 HpyF10VI (MwoI) 124 BstMWI, MwoIm
HpyF3I C↓TNAG FastDigest® DdeI (HpyF3I) 33 HpyF3I (DdeI) 123 BstDEI, DdeIHsp92I GR↓CGYC FastDigest® BsaHI (Hin1I) 24 Hin1I (BsaHI) 120 AcyI, BsaHI, BssNI, BstACIHsp92II CATG↓ FastDigest® NlaIII (Hin1II) 51 Hin1II (NlaIII) 120 [CviAII], FaeI, [FatI], NlaIII
HspAI G↓CGC[FastDigest® HhaI](GCG↓C), FastDigest® HinP1I (Hin6I)
4042
[HhaI](GCG↓C), Hin6I (HinP1I)
120121
[AspLEI], [BstHHI], [CfoI], HinP1I
ItaI GC↓NGC FastDigest® Fnu4HI (SatI)m 38 SatI (Fnu4HI)m 143 Fnu4HIm, Fsp4HIm
KasI G↓GCGCC [FastDigest® EheI](GGC↓GCC) 38[EheI (SfoI)](GGC↓GCC), SspDI (KasI)
117150
[BbeI], [DinI], [EgeI], [Mly113I], [NarI], [SfoIm]
Kpn2I T↓CCGGA FastDigest® Kpn2I 44 Kpn2I (BspEI) 124AccIIIm, Aor13HI, BseAIm, Bsp13I, BspEIm, MroI
KpnI GGTAC↓C[FastDigest® Acc65Im](G↓GTACC), FastDigest® KpnI
1143
[Acc65I (Asp718I)m](G↓GTACC), KpnI
81124
[Asp718Im]
Ksp22I T↓GATCA FastDigest® BclI 20 BclI 87 FbaIKspAI GTT↓AAC FastDigest® HpaI (KspAI) 42 KspAI (HpaI) 125 HpaIKspI CCGC↓GG Cfr42I (SacII) 106 SacII, Sfr303I, SgrBI, SstII
Kzo9I ↓GATCFastDigest® Sau3AI (Bsp143I), FastDigest® MboIm
6044
Bsp143I (Sau3AI), MboIm
99127
BfuCI, BssMI, [BstKTIm], BstMBI, DpnIIm, NdeIIm, Sau3AI
LguI GCTCTTC(1/4)↓ FastDigest® SapI (LguI) 59 LguI (SapI) 125 BspQI, PciSI, SapI
Lsp1109I GCAGC(8/12)↓ FastDigest® BbvI (Lsp1109I), FastDigest® BseXI
1925
Lsp1109I (BbvI), BseXI (BbvI)
12596
BbvI, BstV1I
LweI GCATC(5/9)↓ FastDigest® SfaNI (BmsI) 62 LweI (SfaNI) 126 SfaNIMabI A↓CCWGGT FastDigest® SexAI (CsiI) 61 SexAIm
MaeI C↓TAG FastDigest® BfaI (FspBI) 20 FspBI (BfaI) 119 BfaI, XspIMaeII A↓CGT [FastDigest® TaiI](ACGT↓) 66 [TaiI (MaeII)](ACGT↓) 150 HpyCH4IVMaeIII ↓GTNACMalI GA↓TC FastDigest® DpnI 33 DpnI 108MauBI CG↓CGCGCG FastDigest® MauBI 44 MauBI 126MbiI CCGCTC(-3/-3)↓ FastDigest® BsrBI (MbiI) 30 MbiI (BsrBI) 126 AccBSI, BsrBIm
MboI ↓GATCFastDigest® Sau3AI (Bsp143I)m, FastDigest® MboI
6044
Bsp143I (Sau3AI)m, MboI
99127
BfuCIm, BssMI, [BstKTI], BstMBI, DpnIIm, Kzo9Im, NdeIIm, Sau3AIm
MboII GAAGA(8/7)↓ FastDigest® MboII 45 MboII 127MfeI C↓AATTG FastDigest® MfeI (MunI) 45 MunI (MfeI) 130 MfeIMflI R↓GATCY FastDigest® PsuIm 56 PsuI (BstYI)m 140 BstX2I, BstYIm, XhoIIm
MhlI GDGCH↓C FastDigest® Bsp1286I (SduI) 28 SduI (Bsp1286I) 145 Bsp1286IMlsI TGG↓CCA FastDigest® MscI (MlsI) 47 MlsI (MscI) 128 BalI, MluNI, MscI, Msp20IMluI A↓CGCGT FastDigest® MluI 45 MluI 128MluNI TGG↓CCA FastDigest® MscI (MlsI) 47 MlsI (MscI) 128 BalI, MscI, Msp20I
Mly113I GG↓CGCC [FastDigest® EheI](GGC↓GCC) 38[EheI (SfoI)](GGC↓GCC), [SspDI (KasI)](G↓GCGCC)
117150
[BbeI], [DinI], [EgeI], [KasI], NarI, [SfoI]
MlyI GAGTC(5/5)↓ FastDigest® MlyI (SchI) 46 SchI (MlyI) 144 MlyI, [PleIm], [PpsI]MmeI TCCRAC(20/18)↓MnlI CCTC(7/6)↓ FastDigest® MnlI 46 MnlI 128Mph1103I ATGCA↓T FastDigest® NsiI (Mph1103I) 52 Mph1103I (NsiI) 129 EcoT22I, NsiI, Zsp2IMreI CG↓CCGGCG FastDigest® MreI 46 MreI (Sse232I) 129MroI T↓CCGGA FastDigest® Kpn2I 44 Kpn2I (BspEI) 124 AccIIIm, Aor13HI, BseAIm, Bsp13I, BspEIm
MroNI G↓CCGGC [FastDigest® NaeI (PdiI)](GCC↓GGC) 49 [PdiI (NaeI)](GCC↓GGC) 136 [NaeI], NgoMIVMroXI GAANN↓NNTTC FastDigest® PdmI 53 PdmI (XmnI) 136 Asp700I, XmnIMscI TGG↓CCA FastDigest® MscI (MlsI) 47 MlsI (MscI) 128 BalI, MscI, MluNI, Msp20I
MseI T↓TAAFastDigest® MseI (SaqAI), FastDigest® Tru1I
4767
Tru1I (MseI) 152 MseI, Tru9I
MslI CAYNN↓NNRTG FastDigest® MslI (RseI) 47 RseI (MslI) 142 MslI, SmiMIMsp20I TGG↓CCA FastDigest® MscI (MlsI) 47 MlsI (MscI) 128 BalI, MluNI, MscIMspA1I CMG↓CKGMspCI C↓TTAAG FastDigest® AflII (BspTI) 13 BspTI (AlfII) 101 AflII, BfrI, Bst98I, BstAFI, Vha464I
MspI C↓CGGFastDigest® HpaIIm, FastDigest® MspI
4248
HpaIIm, MspI (HpaII)
122129
BsiSI, HapIIm
MspR9I CC↓NGG FastDigest® ScrFI (Bme1390I) 61 Bme1390I (ScrFI) 91 BmrFI, [BssKI], [BstSCI], ScrFI, [StyD4I]
215
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.25. Commercially available restriction enzymes.
(continued on next page)
Enzyme Specificity 5’→3’ FastDigest® enzyme Page Conventional enzyme PageCommercially available isos-chizomers from other vendors
MssI GTTT↓AAAC FastDigest® MssI 48 MssI (PmeI) 130 PmeIm
MunI C↓AATTG FastDigest® MfeI (MunI) 45 MunI (MfeI) 130 MfeIMva1269I GAATGC(1/-1)↓ FastDigest® Mva1269I 49 Mva1269I (BsmI) 131 BsaMI, BsmI, PctI
MvaI CC↓WGG FastDigest® MvaI 48[EcoRIIm](↓CCWGG), MvaI (BstNI)
116130
[AjnI], BseBI, Bst2UI, BstNI, BstOI, [Psp6I], [PspGIm]
MvnI CG↓CG FastDigest® Bsh1236I 25 Bsh1236I (BstUI) 97 AccII, BspFNI, BstFNI, BstUIMvrI CGAT↓CG FastDigest® PvuI 57 PvuI 141 BpvUI, Ple19IMwoI GCNNNNN↓NNGC FastDigest® HpyF10VIm 124 HpyF10VI (MwoI)m 124 BstMWINaeI GCC↓GGC FastDigest® NaeI (PdiI) 49 PdiI (NaeI) 136 [MroNI], NaeI, [NgoMIV]
NarI GG↓CGCC [FastDigest® EheI](GGC↓GCC) 38[EheI (SfoI)](GGC↓GCC), [SspDI (KasI)](G↓GCGCC)
117150
[BbeI], [DinI], [EgeI], [KasI], Mly113I, [SfoIm]
NciI CC↓SGG FastDigest® NciI (BcnI) 49 BcnI (NciI) 88 AsuC2I, BpuMI, NciINcoI C↓CATGG FastDigest® NcoI 50 NcoI 131 Bsp19INdeI CA↓TATG FastDigest® NdeI 50 NdeI 132 FauNDI
NdeII ↓GATCFastDigest® Sau3AI (Bsp143I)m, FastDigest® MboIm
6044
Bsp143I (Sau3AI)m, MboIm
99127
BfuCIm, BssMI, [BstKTIm], BstMBI, DpnII, Kzo9Im, Sau3AIm
NgoMIV G↓CCGGC [FastDigest® NaeI (PdiI)](GCC↓GGC) 49 [PdiI (NaeI)](GCC↓GGC) 136 MroNI, [NaeI]
NheI G↓CTAGCFastDigest® NheI, [FastDigest® BmtI (BspOI)](GCTAG↓C)
5022
NheI, [BspOI (BmtI)](GCTAG↓C)
132100
AsuNHI, [BmtI]m
NlaIII CATG↓ FastDigest® NlaIII (Hin1II) 51 Hin1II (NlaIII) 120 [CviAII], FaeI, [FatIm], Hsp92II, NlaIIINlaIV GGN↓NCC FastDigest® NlaIV (BspLI)m 51 BspLI (NlaIV)m 100 BmiI, NlaIV, PspN4INmeAIII GCCGAG(21/19)↓NmuCI ↓GTSAC FastDigest® NmuCI 51 NmuCI (Tsp45I) 133 Tsp45INotI GC↓GGCCGC FastDigest® NotI 52 NotI 133 CciNINruI TCG↓CGA FastDigest® NruI (RruI) 52 Bsp68I (NruI) 98 BtuMI, NruINsbI TGC↓GCA FastDigest® FspI (NsbI) 39 NsbI (FspI) 133 Acc16I, AviII, FspINsiI ATGCA↓T FastDigest® NsiI (Mph1103I) 52 Mph1103I (NsiI) 129 EcoT22Im, NsiI, Zsp2INspI RCATG↓Y FastDigest® NspI (XceI) 53 XceI (NspI) 156 BstNSI, NspI
NspV TT↓CGAA FastDigest® Bsp119I 27 Bsp119I (BstBI) 99AsuIIm, Bpu14I, BspT104I, BstBIm, Csp45Im, SfuIm
OliI CACNN↓NNGTG FastDigest® AleI (OliI) 14 OliI (AleI) 134 AleIPacI TTAAT↓TAA FastDigest® PacI 53 PacI 134PaeI GCATG↓C FastDigest® SphI (PaeI) 64 PaeI (SphI) 134 BbuI, SphIPaeR7I C↓TCGAG FastDigest® XhoI 69 XhoI 156 Sfr274I, SlaI, StrI, TliIPagI T↓CATGA FastDigest® BspHI (PagI) 29 PagI (BspHI) 135 CciI, BspHIm, RcaIPalAI GG↓CGCGCC FastDigest® AscI (SgsI) 16 SgsI (AscI) 147 AscIPasI CC↓CWGGG PasI 135PauI G↓CGCGC FastDigest® BssHII (PteI) 31 PauI (BssHII) 135 BsePI, BssHIIPceI AGG↓CCT FastDigest® StuI (Eco147I) 64 Eco147I (StuI) 115 AatI, SseBI, StuIPciI A↓CATGT PscI (PciI) 138PciSI GCTCTTC(1/4)↓ FastDigest® SapI (LguI) 59 LguI (SapI) 125 BspQI, SapIPctI GAATGC(1/-1)↓ FastDigest® Mva1269I 49 Mva1269I (BsmI) 131 BsaMI, BsmIPdiI GCC↓GGC FastDigest® NaeI (PdiI) 49 PdiI (NaeI) 136 [MroNI], NaeI, [NgoMIV]PdmI GAANN↓NNTTC FastDigest® PdmI 53 PdmI (XmnI) 136 Asp700I, MroXI, XmnIm
PfeI G↓AWTC FastDigest® TfiI (PfeI) 67 PfeI (TfiI) 136 TfiIPfl23II G↓GTACG FastDigest® BsiWI (Pfl23II) 26 Pfl23II (BsiWI) 137 BsiWI, PspLIPflFI GACN↓NNGTC FastDigest® PsyI 57 PsyI (Tth111I) 140 AspI, Tth111IPflMI CCANNNN↓NTGG FastDigest® PflMI (Van91I) 54 Van91I (PflMI) 154 AccB7I, BasI, PflMIPfoI T↓CCNGGA FastDigest® PfoI 54 PfoI 137PhoI GG↓CC FastDigest® HaeIII (BsuRI) 40 BsuRI (HaeIII) 103 BshFI, BsnI, BspANI, HaeIIIPinAI A↓CCGGT FastDigest® AgeI (BshTI) 13 BshTI (AgeI) 101 AgeI, AsiGI, CspAIPle19I CGAT↓CG FastDigest® PvuI 57 PvuI 141 BpvUI, MvrIPleI GAGTC(4/5)↓ [FastDigest® MlyI (SchI)](GAGTC(5/5)↓) 46 [SchI (MlyI)](GAGTC(5/5)↓) 144 [MlyIm], PpsIPmaCI CAC↓GTG FastDigest® PmlI (Eco72I) 54 Eco72I (PmlI) 113 AcvI, BbrPI, PmlI, PspCIPmeI GTTT↓AAAC FastDigest® MssIm 48 MssI (PmeI)m 130PmlI CAC↓GTG FastDigest® PmlI (Eco72I) 54 Eco72I (PmlI) 113 AcvI, BbrPI, PmaCI, PmlI, PspCIPpiI ↓(7/12)GAAC(N)5CTC(13/8)↓ PpiI 138PpsI GAGTC(4/5)↓ [FastDigest® MlyI (SchI)](GAGTC(5/5)↓) 46 [SchI (MlyI)](GAGTC(5/5)↓) 144 [MlyI], PleIPpu21I YAC↓GTR FastDigest® BsaAI (Ppu21I) 23 Ppu21I (BsaAI) 138 BsaAI, BstBAIPpuMI RG↓GWCCY FastDigest® PpuMI (Psp5II) 55 Psp5II (PpuMI) 139 PpuMI, PspPPIPscI A↓CATGT PscI (PciI) 138 PciIPshAI GACNN↓NNGTC FastDigest® PshAI (BoxI) 55 BoxI (PshAI) 91 BstPAI, PshAIPshBI AT↓TAAT FastDigest® AseI (VspI) 17 VspI (AseI) 154 AseIPsiI TTA↓TAA FastDigest® PsiI (AanI) 55 AanI (PsiI) 80 PsiI
Psp124BI GAGCT↓C[FastDigest® Ecl136II](GAG↓CTC), FastDigest® SacI
3558
[Ecl136II (EcoICRI)](GAG↓CTC), SacI
109142
[Eco53kI], [EcoICRI], SstI
Psp1406I AA↓CGTT FastDigest® AclI (Psp1406I) 12 Psp1406I (AclI) 139 AclI
216
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.25. Commercially available restriction enzymes.
(continued on next page)
Enzyme Specificity 5’→3’ FastDigest® enzyme Page Conventional enzyme PageCommercially available isos-chizomers from other vendors
Psp5II RG↓GWCCY FastDigest® PpuMI (Psp5II) 55 Psp5II (PpuMI) 139 PpuMI, PspPPI
Psp6I ↓CCWGG [FastDigest® MvaI](CC↓WGG) 48EcoRII, [MvaI (BstNI)](CC↓WGG)
116130
AjnI, [BseBI], [Bst2UI], [BstNI], [BstOI], PspGI
PspCI CAC↓GTG FastDigest® PmlI (Eco72I) 54 Eco72I (PmlI) 113 AcvI, BbrPI, PmaCI, PmlIPspEI G↓GTNACC FastDigest® Eco91I 36 Eco91I (BstEII) 114 BstEII, BstPI, EcoO65IPspFI CCCAGC(-1/-5)↓ FastDigest® PspFI 56 GsaI, [BseYI]
PspGI ↓CCWGG [FastDigest® MvaIm](CC↓WGG) 130EcoRII, [MvaI (BstNI)m](CC↓WGG)
116130
AjnIm, [BseBI], [Bst2UIm], [BstNIm], [BstOIm], Psp6I
PspLI C↓GTACG FastDigest® BsiWI (Pfl23II) 26 Pfl23II (BsiWI) 137 BsiWIPspN4I GGN↓NCC FastDigest® NlaIV (BspLI) 51 BspLI (NlaIV) 100 BmiI, NlaIV
PspOMI G↓GGCCC[FastDigest® ApaI](GGGCC↓C), FastDigest® Bsp120I
1628
[ApaI](GGGCC↓C), Bsp120I (PspOMI)
8599
PspPI G↓GNCC FastDigest® Sau96I (Cfr13I) 60 Cfr13I (Sau96I) 106 AspS9I, [BmgT120I], Sau96IPspPPI RG↓GWCCY FastDigest® PpuMI (Psp5II) 55 Psp5II (PpuMI) 139 PpuMIPspXI VC↓TCGAGBPsrI ↓(7/12)GAAC(N)6TAC(12/7)↓PstI CTGCA↓G FastDigest® PstI 56 PstI 140 BspMAIPsuI R↓GATCY FastDigest® PsuI 56 PsuI (BstYI) 140 BstX2I, BstYI, MflIm, XhoIIPsyI GACN↓NNGTC FastDigest® PsyI 57 PsyI (Tth111I) 140 AspI, PflFI, Tth111IPvuI CGAT↓CG FastDigest® PvuI 57 PvuI 141 BpvUI, MvrI, Ple19IPvuII CAG↓CTG FastDigest® PvuII 57 PvuII 141RcaI T↓CATGA FastDigest® BspHI (PagI) 29 PagI (BspHI) 135 BspHI, CciIRgaI GCGAT↓CGC FastDigest® AsiSI (SfaAI) 17 SfaAI (AsiSI) 145 AsiSI, SgfIRigI GGCCGG↓CC FseI
RsaI GT↓AC[FastDigest® Csp6Im](G↓TAC), FastDigest® RsaI
3258
[Csp6I (CviQI)m](G↓TAC), RsaI
107141
AfaI, [CviQIm], [RsaNI]
RsaNI G↓TACFastDigest® Csp6I, [FastDigest® RsaI](GT↓AC)
3258
Csp6I (CviQI), [RsaI](GT↓AC)
107141
[AfaI], CviQI
RseI CAYNN↓NNRTG FastDigest® MslI (RseI) 47 RseI (MslI) 142 MslI, SmiMIRsr2I CG↓GWCCG FastDigest® RsrII (CpoI) 58 CpoI (RsrII) 106 CspI, RsrIIRsrII CG↓GWCCG FastDigest® RsrII (CpoI) 58 CpoI (RsrII) 106 CspI, Rsr2I, RsrII
SacI GAGCT↓C[FastDigest® Ecl136IIm](GAG↓CTC), FastDigest® SacI
3558
[Ecl136II (EcoICRI)m](GAG↓CTC), SacI
109142
[Eco53kIm], [EcoICRI], Psp124BI, SstI
SacII CCGC↓GG Cfr42I (SacII) 106 KspI, Sfr303I, SgrBI, SstIISalI G↓TCGAC FastDigest® SalI 59 SalI 143SanDI GG↓GWCCC FastDigest® SanDI (KflI) 59 SanDISapI GCTCTTC(1/4)↓ FastDigest® SapI (LguI) 59 LguI (SapI) 125 BspQI, PciSI, SapISatI GC↓NGC FastDigest® Fnu4HI (SatI) 38 SatI (Fnu4HI) 143 Fnu4HI, Fsp4HI, ItaIm
Sau3AI ↓GATCFastDigest® Sau3AI (Bsp143I), FastDigest® MboIm
6044
Bsp143I (Sau3AI), MboIm
99127
BfuCIm, BssMI, [BstKTIm], BstMBI, DpnIIm, Kzo9I, NdeIIm
Sau96I G↓GNCC FastDigest® Sau96I (Cfr13I) 60 Cfr13I (Sau96I) 106 AspS9I, [BmgT120I], PspPI, Sau96ISbfI CCTGCA↓GG FastDigest® SbfI (SdaI) 60 SdaI (SbfI) 144 SbfI, Sse8387IScaI AGT↓ACT FastDigest® ScaI 61 ScaI 144 AssI, BmcAI, ZrmISchI GAGTC(5/5)↓ FastDigest® MlyI (SchI) 46 SchI (MlyI) 144 MlyI, [PleI], [PpsI], SchI
ScrFI CC↓NGG FastDigest® ScrFI (Bme1390I) 61 Bme1390I (ScrFI) 91BmrFI, [BssKI], [BstSCI], MspR9I, ScrFI, [StyD4I]
SdaI CCTGCA↓GG FastDigest® SbfI (SdaI) 60 SdaI (SbfI) 144 SbfI, Sse8387ISduI GDGCH↓C FastDigest® Bsp1286I (SduI) 28 SduI (Bsp1286I) 145 Bsp1286I, MhlISetI ASST↓SexAI A↓CCWGGT FastDigest® SexAI (CsiI)m 61 MabI m, SexAISfaAI GCGAT↓CGC FastDigest® AsiSI (SfaAI) 17 SfaAI (AsiSI) 145 AsiSI, RgaI, SgfISfaNI GCATC(5/9)↓ FastDigest® SfaNI (BmsI) 62 LweI (SfaNI) 126SfcI C↓TRYAG FastDigest® SfcI (BfmI) 62 BfmI (SfcI) 89 BstSFI, SfcISfiI GGCCNNNN↓NGGCC FastDigest® SfiI 62 SfiI 146
SfoI GGC↓GCC FastDigest® EheIm 38EheI (SfoI)m, [SspDI (KasI)](G↓GCGCC)
117150
[BbeIm], DinI, EgeI, [KasIm], [Mly113I], [NarIm]
Sfr274I C↓TCGAG FastDigest® XhoI 69 XhoI 156 PaeR7I, SlaI, StrI, TliISfr303I CCGC↓GG Cfr42I (SacII) 106 KspI, SacII, SgrBI, SstIISgeI m5CNNG(9/13)↓ SgeI 146
SfuI TT↓CGAA FastDigest® Bsp119Im 27 Bsp119I (BstBI)m 99AsuII, Bpu14I, BspT104Im, BstBIm, Csp45I, NspVm
SgfI GCGAT↓CGC FastDigest® AsiSI (SfaAI) 17 SfaAI (AsiSI) 145 AsiSI, RgaISgrAI CR↓CCGGYGSgrBI CCGC↓GG Cfr42I (SacII) 106 KspI, SacII, Sfr303I, SstIISgrDI CG↓TCGACG SgrDI 147SgsI GG↓CGCGCC FastDigest® AscI (SgsI) 16 SgsI (AscI) 147 AscI, PalAISinI G↓GWCC FastDigest® AvaII (Eco47I) 18 Eco47I (AvaII) 111 AvaII, Bme18I, VpaK11BISlaI C↓TCGAG FastDigest® XhoI 69 XhoI 156 PaeR7I, Sfr274I, StrI, TliI
217
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.25. Commercially available restriction enzymes.
(continued on next page)
Enzyme Specificity 5’→3’ FastDigest® enzyme Page Conventional enzyme PageCommercially available isos-chizomers from other vendors
SmaI CCC↓GGG FastDigest® SmaI 63[Cfr9I (XmaI)m](C↓CCGGG), SmaI
105147
[TspMI], [XmaCI], [XmaIm]
SmiI ATTT↓AAAT FastDigest® SwaI (SmiI) 65 SmiI (SwaI) 148 SwaISmiMI CAYNN↓NNRTG FastDigest® MslI (RseI) 47 RseI (MslI) 142 MslISmlI C↓TYRAG SmoI (SmlI) 148SmoI C↓TYRAG SmoI (SmlI) 148 SmlISmuI CCCGC(4/6)↓ SmuI (FauI) 148 FauISnaBI TAC↓GTA FastDigest® SnaBI (Eco105I) 63 Eco105I (SnaBI) 114 BstSNI, SnaBISpeI A↓CTAGT FastDigest® SpeI (BcuI) 63 BcuI (SpeI) 88 AhlI, SpeISphI GCATG↓C FastDigest® SphI (PaeI) 64 PaeI (SphI) 134 BbuI, SphISrfI GCCC↓GGGCSse8387I CCTGCA↓GG FastDigest® SbfI (SdaI) 60 SdaI (SbfI) 144 SbfISse9I ↓AATT FastDigest® Tsp509I (TasI) 68 TasI (Tsp509I) 151 Tsp509I, TspEISseBI AGG↓CCT FastDigest® StuI (Eco147I) 64 Eco147I (StuI) 115 AatI, PceI, StuISsiI CCGC(-3/-1)↓ FastDigest® AciI (SsiI) 12 SsiI (AciI) 149 AciI, BspACI
SspDI G↓GCGCC [FastDigest® EheI](GGC↓GCC) 38[EheI (SfoI)](GGC↓GCC), SspDI (KasI)
117150
[BbeI], [DinI], [EgeI], KasI, [Mly113I], [NarI], [SfoI]
SspI AAT↓ATT FastDigest® SspI 64 SspI 149
SstI GAGCT↓C[FastDigest® Ecl136IIm](GAG↓CTC), FastDigest® SacI
3558
[Ecl136II (EcoICRI)m](GAG↓CTC), SacI
109142
[Eco53kI], [EcoICRI], Psp124BI
SstII CCGC↓GG Cfr42I (SacII) 106 KspI, SacII, Sfr303I, SgrBIStrI C↓TCGAG FastDigest® XhoI 69 XhoI 156 PaeR7I, Sfr274I, SlaI, TliIStuI AGG↓CCT FastDigest® StuI (Eco147I) 64 Eco147I (StuI) 115 AatI, PceI, SseBI, StuIStyD4I ↓CCNGG [FastDigest® ScrFI (Bme1390I)](CC↓NGG) 61 [Bme1390I (ScrFI)](CC↓NGG) 91 [BmrFI], BssKI, BstSCI, [MspR9I], [ScrFI]StyI C↓CWWGG FastDigest® StyI (Eco130I) 65 Eco130I (StyI) 114 BssT1I, EcoT14I, ErhI, StyISwaI ATTT↓AAAT FastDigest® SwaI (SmiI) 65 SmiI (SwaI) 148 SwaITaaI ACN↓GT FastDigest® TaaI 150 TaaI (HpyCH4III) 150 Bst4CI, HpyCH4IIITaiI ACGT↓ FastDigest® TaiI 66 TaiI (MaeII) 150 [HpyCH4IVm], [MaeII]TaqI T↓CGA FastDigest® TaqI 66 TaqI 151
TaqIIGACCGA(11/9)↓,CACCCA(11/9)↓
TasI ↓AATT FastDigest® Tsp509I (TasI) 68 TasI (Tsp509I) 151 Sse9I, Tsp509I, TspEITatI W↓GTACW FastDigest® TatI 66 TatI 151TauI GCSG↓C FastDigest® TauI 67 TauI 152TfiI G↓AWTC FastDigest® TfiI (PfeI) 67 PfeI (TfiI) 136 TfiITliI C↓TCGAG FastDigest® XhoI 69 XhoI 156 PaeR7I, Sfr274I, SlaI, StrI
Tru1I T↓TAAFastDigest® MseI (SaqAI), FastDigest® Tru1I
4767
Tru1I (MseI) 152 MseI, Tru9I
Tru9I T↓TAAFastDigest® MseI (SaqAI), FastDigest® Tru1I
4767
Tru1I (MseI) 152 MseI
TscAI CASTG(2/-7)↓ FastDigest® TspRI (TscAI) 68 TscAI (TspRI) 153 TspRITseI G↓CWGC ApeKITsoI TARCCA(11/9)↓ TsoI 153Tsp45I ↓GTSAC FastDigest® NmuCI 51 NmuCI (Tsp45I) 133Tsp509I ↓AATT FastDigest® Tsp509I (TasI) 68 TasI (Tsp509I) 151 Sse9I, Tsp509I, TspEITspDTI ATGAA(11/9)↓TspEI ↓AATT FastDigest® Tsp509I (TasI) 68 TasI (Tsp509I) 151 Sse9I, Tsp509ITspGWI ACGGA(11/9)↓
TspMI C↓CCGGG [FastDigest® SmaI](CCC↓GGG) 63Cfr9I (XmaI)m, [SmaI](CCC↓GGG)
105147
XmaCI, XmaIm
TspRI CASTGNN(2/-7)↓ FastDigest® TspRI (TscAI) 68 TscAI (TspRI) 153 TspRITstI ↓(8/13)CAC(N)6TCC(12/7)↓ TstI 154Tth111I GACN↓NNGTC FastDigest® PsyI 57 PsyI (Tth111I) 140 AspI, PflFIVan91I CCANNNN↓NTGG FastDigest® PflMI (Van91I) 54 Van91I (PflMI) 154 AccB7I, BasI, PflMIVha464I C↓TTAAG FastDigest® AflII (BspTI) 13 BspTI (AflII) 101 AflII, BfrI, Bst98I, BstAFI, MspCIVneI G↓TGCAC FastDigest® ApaLI (Alw44I) 16 Alw44I (ApaLI) 85 ApaLIVpaK11BI G↓GWCC FastDigest® AvaII (Eco47I) 18 Eco47I (AvaII) 111 AvaII, Bme18I, SinIVspI AT↓TAAT FastDigest® AseI (VspI) 17 VspI (AseI) 154 AseI, PshBIXagI CCTNN↓NNNAGG FastDigest® EcoNI (XagI) 36 XagI (EcoNI) 155 BstENI, EcoNIXapI R↓AATTY FastDigest® XapI 68 XapI (ApoI) 155 AcsI, ApoIXbaI T↓CTAGA FastDigest® XbaI 69 XbaI 155XceI RCATG↓Y FastDigest® NspI (XceI) 53 XceI (NspI) 156 BstNSI, NspIXcmI CCANNNNN↓NNNNTGGXhoI C↓TCGAG FastDigest® XhoI 69 XhoI 156 PaeR7I, Sfr274I, SlaI, StrI, TliIXhoII R↓GATCY FastDigest® PsuI 56 PsuI (BstYI) 140 BstX2I, BstYI, MflIm,
XmaCI C↓CCGGG [FastDigest® SmaI](CCC↓GGG) 63Cfr9I (XmaI), [SmaI](CCC↓GGG)
105147
TspMI, XmaI
218
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.25. Commercially available restriction enzymes.
Enzyme Specificity 5’→3’ FastDigest® enzyme Page Conventional enzyme PageCommercially available isos-chizomers from other vendors
XmaI C↓CCGGG [FastDigest® SmaIm](CCC↓GGG) 63Cfr9I (XmaI)m, [SmaIm](CCC↓GGG)
105147
TspMIm, XmaCI
XmaJI C↓CTAGG FastDigest® AvrII (XmaJI) 18 XmaJI (AvrII) 156 AspA2I, AvrII, BlnIXmiI GT↓MKAC FastDigest® AccI (XmiI) 11 XmiI (AccI) 157 AccI, FblIXmnI GAANN↓NNTTC FastDigest® PdmIm 53 PdmI (XmnI)m 136 Asp700I, MroXIXspI C↓TAG FastDigest® BfaI (FspBI) 20 FspBI (BfaI) 119 BfaI, MaeIZraI GAC↓GTC [FastDigest® AatIIm](GACGT↓C) 11 [AatIIm](GACGT↓C) 81ZrmI AGT↓ACT FastDigest® ScaI 144 ScaI 144 AssI, BmcAIZsp2I ATGCA↓T FastDigest® NsiI (Mph1103I) 52 Mph1103I (NsiI) 129 EcoT22I, NsiI
219
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.26. Recognition sequences of restriction enzymes.
Alphabetic List of Recognition Sequences
(continued on next page)
Specificity 5’→3’
FastDigest® enzyme
Conventional enzyme
AA↓CGTT FastDigest® AclI (Psp1406I) Psp1406I (AclI)A↓AGCTT FastDigest® HindIII HindIII↓(8/13)AAG(N)5CTT(13/8)↓ FalIAAT↓ATT FastDigest® SspI SspI↓AATT FastDigest® Tsp509I (TasI) TasI (Tsp509I)A↓CATGT PscI (PciI)A↓CCGGT FastDigest® AgeI (BshTI) BshTI (AgeI)ACCTGC(4/8)↓* FastDigest® BspMI (BveI) BveI (BspMI)A↓CCWGGT FastDigest® SexAI (CsiI) SexAIA↓CGCGT FastDigest® MluI MluIACGGA(11/9)↓* TspGWIACGGC(12/14)↓* BceAIACGT↓ FastDigest® TaiI TaiI (MaeII)A↓CGT MaeIIACN↓GT FastDigest® TaaI TaaI (HpyCH4III)↓(10/15)AC(N)4GTAYC(12/7)↓* BaeI↓(9/12)AC(N)5CTCC(10/7)↓* BsaXIA↓CRYGT AflIIIA↓CTAGT FastDigest® SpeI (BcuI) BcuI (SpeI)ACTGG(1/-1)↓* FastDigest® BseNI BseNI (BsrI)ACTGGG(5/4)↓* BfiI (BmrI)A↓GATCT FastDigest® BglII BglIIAGC↓GCT FastDigest® AfeI (Eco47III) Eco47III (AfeI)AG↓CT FastDigest® AluI AluIAGG↓CCT FastDigest® StuI (Eco147I) Eco147I (StuI)AGT↓ACT FastDigest® ScaI ScaIASST↓ SetIAT↓CGAT FastDigest® ClaI (Bsu15I) Bsu15I (ClaI)ATGAA(11/9)↓* TspDTIATGCA↓T FastDigest® NsiI (Mph1103I) Mph1103I (NsiI)AT↓TAAT FastDigest® AseI (VspI) VspI (AseI)ATTT↓AAAT FastDigest® SwaI (SmiI) SmiI (SwaI)C↓(11/13)CAA(N)5GTGG(12/10)↓* CspCIC↓AATTG FastDigest® MfeI (MunI) MunI (MfeI)CACCCA(11/9)↓* TaqIICACCTGC(4/8)↓* AarICACGAG(-5/-1)↓* BauI (BssSI)CACGTC(-3/-3)↓* AjiI (BmgBI)CAC↓GTG FastDigest® PmlI (Eco72I) Eco72I (PmlI)CACNNN↓GTG FastDigest® DraIII (AdeI) AdeI (DraIII)CACNN↓NNGTG FastDigest® AleI (OliI) OliI (AleI)↓(8/13)CAC(N)6TCC(12/7)↓* TstI↓(0/2)CACTGC* BtsICAGCAG(25/27)↓* EcoP15ICAG↓CTG FastDigest® PvuII PvuIICAGNNN↓CTG FastDigest® AlwNI (CaiI) CaiI (AlwNI)CASTG(2/-7)↓ FastDigest® TspRI (TscAI) TscAI (TspRI)CA↓TATG FastDigest® NdeI NdeI↓(13/9)CATCC* FastDigest® FokI FokI↓(0/2)CATCC* FastDigest® BseGI BseGI (BtsCI)↓(14/10)CATCGC* BtgZICATG↓ FastDigest® NlaIII (Hin1II) Hin1II (NlaIII)↓CATG FatIC↓ATG CviAII↓(0/2)CATTGC* FastDigest® BsrDI (BseMI) BseMI (BsrDI)CAYNN↓NNRTG FastDigest® MslI (RseI) RseI (MslI)↓(6/11)CCAA(N)7TTC(12/7)↓* FastDigest® AjuI AjuI↓(10/12)CCAC(N)5TTG(13/11)↓* CspCI
Specificity 5’→3’
FastDigest® enzyme
Conventional enzyme
↓(-1/1)CCAGT* FastDigest® BseNI BseNI (BsrI)CCANNNNN↓NNNNTGG XcmICCANNNNN↓NTGG FastDigest® BstXI BstXICCANNNN↓NTGG FastDigest® PflMI (Van91I) Van91I (PflMI)CCATC(4/5)↓* BccIC↓CATGG FastDigest® NcoI NcoICCCAGC(-5/-1)↓* BseYICCCAGC(-1/-5)↓* FastDigest® PspFI GsaI↓(4/5)CCCAGT* BfiI (BmrI)CCCGC(4/6)↓* SmuI (FauI)C↓CCGGG Cfr9I (XmaI)CCC↓GGG FastDigest® SmaI SmaICC↓CWGGG PasICCGC(-3/-1)↓* FastDigest® AciI (SsiI) SsiI (AciI)CCGC↓GG Cfr42I (SacII)CCGCTC(-3/-3)↓* FastDigest® BsrBI (MbiI) MbiI (BsrBI)
C↓CGGFastDigest® HpaIIFastDigest® MspI
HpaIIMspI (HpaII)
↓CCNGG StyD4ICC↓NGG FastDigest® ScrFI (Bme1390I) Bme1390I (ScrFI)C↓CNNGG FastDigest® BsaJI (BseDI) BseDI (BsaJI)CCNNNNN↓NNGG FastDigest® BslI (BseLI) BseLI (BslI)C↓CRYGG BtgICC↓SGG FastDigest® NciI (BcnI) BcnI (NciI)C↓CTAGG FastDigest® AvrII (XmaJI) XmaJI (AvrII)CCTC(7/6)↓* FastDigest® MnlI MnlICCTCAGC(-5/-2)↓* BbvCICC↓TCGAGG AbsICCTGCA↓GG FastDigest® SbfI (SdaI) SdaI (SbfI)CCTNAGC(-5/-2)↓* FastDigest® Bpu10I Bpu10ICC↓TNAGG FastDigest® Bsu36I (Eco81I) Eco81I (Bsu36I)CCTNN↓NNNAGG FastDigest® EcoNI (XagI) XagI (EcoNI)CCTTC(6/5)↓* HpyAV↓CCWGG EcoRIICC↓WGG FastDigest® MvaI MvaI (BstNI)C↓CWWGG FastDigest® StyI (Eco130I) Eco130I (StyI)↓(10/12)CGA(N)6TGC(12/10)↓* BcgICGAT↓CG FastDigest® PvuI PvuICG↓CCGGCG FastDigest® MreI MreI (Sse232I)CG↓CG FastDigest® Bsh1236I Bsh1236I (BstUI)CG↓CGCGCG FastDigest® MauBI MauBIC↓GGCCG FastDigest® EagI (Eco52I) Eco52I (EagI)CG↓GWCCG FastDigest® RsrII (CpoI) CpoI (RsrII)CGRY↓CG FastDigest® BsiEI (Bsh1285I) Bsh1285I (BsiEI)C↓GTACG FastDigest® BsiWI (Pfl23II) Pfl23II (BsiWI)CG↓TCGACG SgrDICGTCTC(1/5)↓* FastDigest® BsmBI (Esp3I) Esp3I (BsmBI)CGWCG↓ Hpy99ICMG↓CKG MspA1Im5CNNG(9/13)↓ SgeI↓(6/11)CRAA(N)6GTC(13/18)↓* ArsICR↓CCGGYG SgrAIC↓TAG FastDigest® BfaI (FspBI) FspBI (BfaI)↓(14/16)CTCAAG* BpuEICTCAG(9/7)↓* BspCNICTCAG(10/8)↓* FastDigest® BspCNI (BseMII) BseMII (BspCNI)↓(14/16)CTCCAG* FastDigest® BpmI (GsuI) GsuI (BpmI)↓(8/10)CTCCTC* BseRIC↓TCGAG FastDigest® XhoI XhoI
220
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.26. Recognition sequences of restriction enzymes.
(continued on next page)
Specificity 5’→3’
FastDigest® enzyme
Conventional enzyme
↓(19/21)CTCGGC* NmeAIIICTCGTG(-5/-1)↓* BauI (BssSI)CTCTTC(1/4)↓* FastDigest® EarI (Eam1104I) Eam1104I (EarI)CTGAAG(16/14)↓* FastDigest® AcuI (Eco57I) Eco57I (AcuI)↓(7/9)CTGAG* BspCNI↓(8/10)CTGAG* FastDigest® BspCNI (BseMII) BseMII (BspCNI)↓(14/16)CTGCAC* BsgICTGCA↓G FastDigest® PstI PstI↓(27/25)CTGCTG* EcoP15ICTGGAG(16/14)↓* FastDigest® BpmI (GsuI) GsuI (BpmI)CTGRAG(16/14)↓* Eco57MIC↓TNAG FastDigest® DdeI (HpyF3I) HpyF3I (DdeI)C↓TRYAG FastDigest® SfcI (BfmI) BfmI (SfcI)C↓TTAAG FastDigest® AflII (BspTI) BspTI (AflII)↓(14/16)CTTCAG* FastDigest® AcuI (Eco57I) Eco57I (AcuI)CTTGAG(16/14)↓* BpuEI↓(14/16)CTYCAG* Eco57MIC↓TYRAG SmoI (SmlI)C↓YCGRG FastDigest® AvaI (Eco88I) Eco88I (AvaI)CY↓CGRG BmeT110IG↓(7/12)GAAC(N)5CTC(13/8)↓* PpiI↓(7/12)GAAC(N)6TAC(12/7)↓* PsrI↓(7/12-13)GAAC(N)6TCC(12-13/7)↓* AloIGAAGA(8/7)↓* FastDigest® MboII MboIIGAAGAC(2/6)↓* FastDigest® BbsI (BpiI) BpiI (BbsI)↓(4/1)GAAGAG* FastDigest® EarI (Eam1104I) Eam1104I (EarI)↓(4/1)GAAGAGC* FastDigest® SapI (LguI) LguI (SapI)↓(5/6)GAAGG* HpyAV↓(7/12)GAAG(N)6TAC(12/7)↓* BarI↓(7/12)GAA(N)7TTGG(11/6)↓* FastDigest® AjuI AjuIGAANN↓NNTTC FastDigest® PdmI PdmI (XmnI)GAATGC(1/-1)↓* FastDigest® Mva1269I Mva1269I (BsmI)G↓AATTC FastDigest® EcoRI EcoRI↓(8/13-14)GAB(N)5RTC(13-14/8)↓* Hin4IGACCGA(11/9)↓* TaqIIGACGC(5/10)↓* FastDigest® HgaI (CseI) CseI (HgaI)GAC↓GTC ZraIGACGT↓C FastDigest® AatII AatIIGACGTG(-3/-3)↓* AjiI (BmgBI)GACN↓NNGTC FastDigest® PsyI PsyI (Tth111I)GACNN↓NNGTC FastDigest® PshAI (BoxI) BoxI (PshAI)GACNNN↓NNGTC FastDigest® Eam1105I Eam1105I (AhdI)GACNNNN↓NNGTC FastDigest® DrdI (AasI) AasI (DrdI)↓(8/13)GAC(N)6TTYG(11/6)↓* ArsI↓(5/5)GACTC* FastDigest® MlyI (SchI) SchI (MlyI)↓(5/4)GACTC* PleI↓(5/1)GAGAC* FastDigest® Alw26I Alw26I (BsmAI)↓(5/1)GAGACC* FastDigest® Eco31I Eco31I (BsaI)↓(5/1)GAGACG* FastDigest® BsmBI (Esp3I) Esp3I (BsmBI)GAGCGG(-3/-3)↓* FastDigest® BsrBI (MbiI) MbiI (BsrBI)GAGCT↓C FastDigest® SacI SacIGAG↓CTC FastDigest® Ecl136II Ecl136II (EcoICRI)↓(6/7)GAGG* FastDigest® MnlI MnlIGAGGAG(10/8)↓* BseRI↓(8/13)GAG(N)5CTC(13/8)↓ FastDigest® BplI BplI↓(8/13)GAG(N)5GTTC(12/7)↓* PpiIGAGTC(5/5)↓* FastDigest® MlyI (SchI) SchI (MlyI)GAGTC(4/5)↓* PleIG↓ANTC FastDigest® HinfI HinfIGAT↓ATC FastDigest® EcoRV (Eco32I) Eco32I (EcoRV)
Specificity 5’→3’
FastDigest® enzyme
Conventional enzyme
↓GATCFastDigest® Sau3AI (Bsp143I)FastDigest® MboI
Bsp143I (Sau3AI)MboI
Gm6A↓TC FastDigest® DpnI DpnIGAT↓C BstKTI↓(5/4)GATCC* BspPI (AlwI)↓(9/5)GATGC* FastDigest® SfaNI (BmsI) LweI (SfaNI)↓(5/4)GATGG* BccIGATNN↓NNATC FastDigest® BsaBI (BseJI) BseJI (BsaBI)G↓AWTC FastDigest® TfiI (PfeI) PfeI (TfiI)↓(8/13-14)GAY(N)5VTC(13-14/8)↓* Hin4IGCAATG(2/0)↓* FastDigest® BsrDI (BseMI) BseMI (BsrDI)
GCAGC(8/12)↓*FastDigest® BbvI (Lsp1109I)FastDigest® BseXI
Lsp1109I (BbvI)BseXI (BbvI)
↓(8/4)GCAGGT* FastDigest® BspMI (BveI) BveI (BspMI)↓(8/4)GCAGGTG* AarIGCAGTG(2/0)↓* BtsI↓(10/12)GCA(N)6TCG(12/10)↓* BcgI↓(10/12)GCA(N)6TGC(12/10)↓ AlfIGCANNNN↓NTGC BstAPIGCATC(5/9)↓* FastDigest® SfaNI (BmsI) LweI (SfaNI)GCATG↓C FastDigest® SphI (PaeI) PaeI (SphI)↓(-1/1)GCATTC* FastDigest® Mva1269I Mva1269I (BsmI)GCCC↓GGGC SrfIGCCGAG(21/19)↓* NmeAIIIG↓CCGGC NgoMIVGCC↓GGC FastDigest® NaeI (PdiI) PdiI (NaeI)↓(14/12)GCCGT* BceAIGCCNNNN↓NGGC FastDigest® BglI BglIGCGAT↓CGC FastDigest® AsiSI (SfaAI) SfaAI (AsiSI)GCGATG(10/14)↓* BtgZIG↓CGC FastDigest® HinP1I (Hin6I) Hin6I (HinP1I)GCG↓C FastDigest® HhaI HhaIGC↓CG GlaIG↓CGCGC FastDigest® BssHII (PteI) PauI (BssHII)GCGG(-3/-1)↓* FastDigest® AciI (SsiI) SsiI (AciI)GC↓GGCCGC FastDigest® NotI NotI↓(6/4)GCGGG* SmuI (FauI)↓(10/5)GCGTC* FastDigest® HgaI (CseI) CseI (HgaI)GC↓NGC FastDigest® Fnu4HI (SatI) SatI (Fnu4HI)GC↓NGC BisIGCN↓GC BlsIGCN↓NGC Cac8IGCNNNNN↓NNGC FastDigest® HpyF10VI HpyF10VI (MwoI)GCSG↓C FastDigest® TauI TauIGCTAG↓C FastDigest® BmtI (BspOI) BspOI (BmtI)G↓CTAGC FastDigest® NheI NheIGCTCTTC(1/4)↓* FastDigest® SapI (LguI) LguI (SapI)GCTGAGG(-5/-2)↓* BbvCI
↓(12/8)GCTGC*FastDigest® BbvI (Lsp1109I)FastDigest® BseXI
Lsp1109I (BbvI)BseXI (BbvI)
GCTGGG(-5/-1)↓* BseYIGCTGGG(-1/-5)↓* FastDigest® PspFI GsaIGC↓TNAGC FastDigest® BlpI (Bpu1102I) Bpu1102I (BlpI)GCTNAGG(-5/-2)↓* FastDigest® Bpu10I Bpu10IG↓CWGC TseIGDGCH↓C FastDigest® Bsp1286I (SduI) SduI (Bsp1286I)↓(7/10)GGAG(N)5GT(12/9)↓* BsaXI↓(7/12)GGA(N)6GTG(13/8)↓* TstI↓(7/12-13)GGA(N)6GTTC(12-13/7)↓* AloI↓(5/6)GGATAC* BfuI (BciVI)GGATC(4/5)↓* BspPI (AlwI)
221
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.26. Recognition sequences of restriction enzymes.
Note* Asymetric sequences.• Fermentas FastDigest® enzymes are shown in ruby.• Fermentas conventional enzymes are shown in orange.
Single letter codeR = G or A; K = G or T; B = C, G or T;Y = C or T; S = C or G; D = A, G or T;W = A or T; H = A, C or T; N = G, A, T or C.M = A or C; V = A, C or G;
Specificity 5’→3’
FastDigest® enzyme
Conventional enzyme
G↓GATCC FastDigest® BamHI BamHIGGATG(9/13)↓* FastDigest® FokI FokIGGATG(2/0)↓* FastDigest® BseGI BseGI (BtsCI)GG↓CC FastDigest® HaeIII (BsuRI) BsuRI (HaeIII)GGCCGG↓CC FseIGGCCNNNN↓NGGCC FastDigest® SfiI SfiIGGC↓GCC FastDigest® EheI EheI (SfoI)GG↓CGCC NarIG↓GCGCC SspDI (KasI)GGCGC↓C BbeIGG↓CGCGCC FastDigest® AscI (SgsI) SgsI (AscI)GGCGGA(11/9)↓* EciIGGGAC(10/14)↓* FastDigest® BsmFI (FaqI) FaqI (BsmFI)G↓GGCCC FastDigest® Bsp120I Bsp120I (PspOMI)GGGCC↓C FastDigest® ApaI ApaIGG↓GWCCC FastDigest® SanDI (KflI) SanDIG↓GNCC FastDigest® Sau96I (Cfr13I) Cfr13I (Sau96I)GG↓NCC BmgT120IGGN↓NCC FastDigest® NlaIV (BspLI) BspLI (NlaIV)GGTAC↓C FastDigest® KpnI KpnIG↓GTACC FastDigest® Acc65I Acc65I (Asp718I)GGTCTC(1/5)↓* FastDigest® Eco31I Eco31I (BsaI)GGTGA(8/7)↓* HphIG↓GTNACC FastDigest® Eco91I Eco91I (BstEII)G↓GWCC FastDigest® AvaII (Eco47I) Eco47I (AvaII)G↓GYRCC FastDigest® BanI (BshNI) BshNI (BanI)GKGCM↓C FastDigest® Bme1580I (BseSI) BseSI (Bme1580I)GR↓CGYC FastDigest® BsaHI (Hin1I) Hin1I (BsaHI)GRGCY↓C Eco24I (BanII)↓(7/12)GRTAC(N)4GT(15/10)↓* BaeIGT↓AC FastDigest® RsaI RsaIG↓TAC FastDigest® Csp6I Csp6I (CviQI)↓(7/12)GTA(N)6CTTC(12/7)↓* BarI↓(7/12)GTA(N)6GTTC(12/7)↓* PsrIGTA↓TAC FastDigest® BstZ17I (Bst1107I) Bst1107I (BstZ17I)GTATCC(6/5)↓* BfuI (BciVI)↓(14/10)GTCCC* FastDigest® BsmFI (FaqI) FaqI (BsmFI)G↓TCGAC FastDigest® SalI SalIGTCTC(1/5)↓* FastDigest® Alw26I Alw26I (BsmAI)↓(6/2)GTCTTC* FastDigest® BbsI (BpiI) BpiI (BbsI)G↓TGCAC FastDigest® ApaLI (Alw44I) Alw44I (ApaLI)GTGCAG(16/14)↓* BsgIGT↓MKAC FastDigest® AccI (XmiI) XmiI (AccI)↓GTNAC MaeIIIGTN↓NAC FastDigest® Hpy8I Hpy8I (MjaIV)↓GTSAC FastDigest® NmuCI NmuCI (Tsp45I)GTT↓AAC FastDigest® HpaI (KspAI) KspAI (HpaI)GTTT↓AAAC FastDigest® MssI MssI (PmeI)↓(18/20)GTYGGA* MmeIGTY↓RAC FastDigest® HincII HincII (HindII)GWGCW↓C FastDigest® Alw21I Alw21I (BsiHKAI)RR↓AATTY FastDigest® XapI XapI (ApoI)RCATG↓Y FastDigest® NspI (XceI) XceI (NspI)R↓CCGGY FastDigest® BsrFI (Cfr10I) Cfr10I (BsrFI)R↓GATCY FastDigest® PsuI PsuI (BstYI)RGCGC↓Y FastDigest® HaeII (BfoI) HaeIIRG↓CY CviJIRG↓GNCCY FastDigest® EcoO109I EcoO109I (DraII)RG↓GWCCY FastDigest® PpuMI (Psp5II) Psp5II (PpuMI)RTGC↓GCAY FastDigest® FspAI FspAI
Specificity 5’→3’
FastDigest® enzyme
Conventional enzyme
TTAC↓GTA FastDigest® SnaBI (Eco105I) Eco105I (SnaBI)TARCCA(11/9)↓* TsoI↓(7/8)TCACC* HphIT↓CATGA FastDigest® BspHI (PagI) PagI (BspHI)↓(9/11)TCCGCC* EciIT↓CCGGA FastDigest® Kpn2I Kpn2I (BspEI)↓(9/11)TCCGT* TspGWI↓(9/11)TCGGTC* TaqIIT↓CCNGGA FastDigest® PfoI PfoITCCRAC(20/18)↓* MmeIT↓CGA FastDigest® TaqI TaqITCG↓CGA FastDigest® NruI (RruI) Bsp68I (NruI)TCN↓GA Hpy188ITC↓NNGA Hpy188IIIT↓CTAGA FastDigest® XbaI XbaI↓(7/8)TCTTC* FastDigest® MboII MboII↓(10/12)TGA(N)6TCA(12/10)↓ BdaIT↓GATCA FastDigest® BclI BclITG↓CA HpyCH4VTGC↓GCA FastDigest® FspI (NsbI) NsbI (FspI)TGG↓CCA FastDigest® MscI (MlsI) MlsI (MscI)↓(9/11)TGGGTG* TaqII↓(9/11)TGGYTA* FastDigest® MscI (MlsI) TsoIT↓GTACA FastDigest® Bsp1407I Bsp1407I (BsrGI)
T↓TAAFastDigest® MseI (SaqAI)FastDigest® Tru1I
Tru1I (MseI)
TTAAT↓TAA FastDigest® PacI PacITTA↓TAA FastDigest® PsiI (AanI) AanI (PsiI)↓(9/11)TTCAT* TspDTITT↓CGAA FastDigest® Bsp119I Bsp119I (BstBI)TTS↓AA AgsITTT↓AAA FastDigest® DraI DraIVVC↓TCGAGB PspXIWW↓CCGGW BsaWIW↓GTACW FastDigest® TatI TatIYYAC↓GTR FastDigest® BsaAI (Ppu21I) Ppu21I (BsaAI)YA↓TR FaiIY↓GGCCR CfrI (EaeI)
222
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Enzyme Recognizing 2 bp Long TargetsTable 1.27. Enzyme recognizing 2 bp long targets.
Alphabetic List of Enzymes Recognizing 5 bp Long Targets
Note* Asymetric sequences.• Fermentas FastDigest® enzymes are shown in ruby.• Fermentas conventional enzymes are shown in orange.
Single letter codeR = G or A; K = G or T; B = C, G or T;Y = C or T; S = C or G; D = A, G or T;W = A or T; H = A, C or T; N = G, A, T or C.M = A or C; V = A, C or G;
Specificity 5’→3’
FastDigest® enzyme
Conventional enzyme
m5CNNG(9/13)↓ SgeI
Alphabetic List of Enzymes Recognizing 4 bp Long TargetsTable 1.28. Enzymes recognizing 4 bp long targets.Specificity 5’→3’
FastDigest® enzyme
Conventional enzyme
↓AATT FastDigest® Tsp509I (TasI) TasI (Tsp509I)ACGT↓ FastDigest® TaiI TaiI (MaeII)A↓CGT MaeIIACN↓GT FastDigest® TaaI TaaI (HpyCH4III)AG↓CT FastDigest® AluI AluIASST↓ SetICATG↓ FastDigest® NlaIII (Hin1II) Hin1II (NlaIII)↓CATG FatIC↓ATG CviAII CCGC(-3/-1)↓* FastDigest® AciI (SsiI) SsiI (AciI)
C↓CGGFastDigest® HpaIIFastDigest® MspI
HpaIIMspI (HpaII)
↓CCNGG StyD4ICC↓NGG FastDigest® ScrFI (Bme1390I) Bme1390I (ScrFI)C↓CNNGG FastDigest® BsaJI (BseDI) BseDI (BsaJI)CCNNNNN↓NNGG FastDigest® BslI (BseLI) BseLI (BslI)CCTC(7/6)↓* FastDigest® MnlI MnlICG↓CG FastDigest® Bsh1236I Bsh1236I (BstUI)C↓TAG FastDigest® BfaI (FspBI) FspBI (BfaI)C↓TNAG FastDigest® DdeI (HpyF3I) HpyF3I (DdeI)↓(6/7)GAGG* FastDigest® MnlI MnlIG↓ANTC FastDigest® HinfI HinfI
↓GATCFastDigest® MboIFastDigest® Sau3AI (Bsp143I)
MboIBsp143I (Sau3AI)
Gm6A↓TC FastDigest® DpnI DpnIGAT↓C BstKTIG↓CGC FastDigest® HinP1I (Hin6I) Hin6I (HinP1I)GCG↓C FastDigest® HhaI HhaIGC↓GC GlaIGCGG(-3/-1)↓* FastDigest® AciI (SsiI) SsiI (AciI)GC↓NGC FastDigest® Fnu4HI (SatI) SatI (Fnu4HI)GC↓NGC BisIGCN↓GC BlsIGCN↓NGC Cac8IGCNNNNN↓NNGC FastDigest® HpyF10VI HpyF10VI (MwoI)GG↓CC FastDigest® HaeIII (BsuRI) BsuRI (HaeIII)G↓GNCC FastDigest® Sau96I (Cfr13I) Cfr13I (Sau96I)GG↓NCC BmgT120IGGN↓NCC FastDigest® NlaIV (BspLI) BspLI (NlaIV)GT↓AC FastDigest® RsaI RsaIG↓TAC FastDigest® Csp6I Csp6I (CviQI)↓GTNAC MaeIIIGTN↓NAC FastDigest® Hpy8I Hpy8I (MjaIV)RG↓CY CviJIT↓CGA FastDigest® TaqI TaqITCN↓GA Hpy188ITC↓NNGA Hpy188IIITG↓CA HpyCH4V
T↓TAAFastDigest® MseI (SaqAI)FastDigest® Tru1I
Tru1I (MseI)
YA↓TR FaiI
Specificity 5’→3’
FastDigest® enzyme
Conventional enzyme
ACGGA(11/9)↓* TspGWIACGGC(12/14)↓* BceAIACTGG(1/-1)↓* FastDigest® BseNI BseNI (BsrI)ATGAA(11/9)↓* TspDTICASTG(2/-7)↓ FastDigest® TspRI (TscAI) TscAI (TspRI)↓(13/9)CATCC* FastDigest® FokI FokI↓(0/2)CATCC* FastDigest® BseGI BseGI (BtsCI)↓(-1/1)CCAGT* FastDigest® BseNI BseNI (BsrI)CCATC(4/5)↓* BccICCCGC(4/6)↓* SmuI (FauI)CC↓SGG FastDigest® NciI (BcnI) BcnI (NciI)CCTTC(6/5)↓* HpyAV↓CCWGG EcoRIICC↓WGG FastDigest® MvaI MvaI (BstNI)CGWCG↓ Hpy99ICTCAG(9/7)↓* BspCNICTCAG(10/8)↓* FastDigest® BspCNI (BseMII) BseMII (BspCNI)↓(7/9)CTGAG* BspCNI↓(8/10)CTGAG* FastDigest® BspCNI (BseMII) BseMII (BspCNI)GAAGA(8/7)↓* FastDigest® MboII MboII↓(5/6)GAAGG* HpyAVGACGC(5/10)↓* FastDigest® HgaI (CseI) CseI (HgaI)↓(5/5)GACTC* FastDigest® MlyI (SchI) SchI (MlyI)↓(5/4)GACTC* PleI↓(5/1)GAGAC* FastDigest® Alw26I Alw26I (BsmAI)GAGTC(5/5)↓* FastDigest® MlyI (SchI) SchI (MlyI)GAGTC(4/5)↓* PleI↓(5/4)GATCC* BspPI (AlwI)↓(9/5)GATGC* FastDigest® SfaNI (BmsI) LweI (SfaNI)↓(5/4)GATGG* BccIG↓AWTC FastDigest® TfiI (PfeI) PfeI (TfiI)
GCAGC(8/12)↓*FastDigest® BbvI (Lsp1109I)FastDigest® BseXI
Lsp1109I (BbvI)BseXI (BbvI)
GCATC(5/9)↓* FastDigest® SfaNI (BmsI) LweI (SfaNI)↓(14/12)GCCGT* BceAI↓(6/4)GCGGG* SmuI (FauI)↓(10/5)GCGTC* FastDigest® HgaI (CseI) CseI (HgaI)GCSG↓C FastDigest® TauI TauI
↓(12/8)GCTGC*FastDigest® BbvI (Lsp1109I)FastDigest® BseXI
Lsp1109I (BbvI)BseXI (BbvI)
G↓CWGC TseIGGATC(4/5)↓* BspPI (AlwI)GGATG(9/13)↓* FastDigest® FokI FokIGGATG(2/0)↓* FastDigest® BseGI BseGI (BtsCI)GGGAC(10/14)↓* FastDigest® BsmFI (FaqI) FaqI (BsmFI)GGTGA(8/7)↓* HphIG↓GWCC FastDigest® AvaII (Eco47I) Eco47I (AvaII)↓(14/10)GTCCC* FastDigest® BsmFI (FaqI) FaqI (BsmFI)GTCTC(1/5)↓* FastDigest® Alw26I Alw26I (BsmAI)↓GTSAC FastDigest® NmuCI NmuCI (Tsp45I)↓(7/8)TCACC* HphI↓(9/11)TCCGT* TspGWI↓(7/8)TCTTC* FastDigest® MboII MboII↓(9/11)TTCAT* TspDTITTS↓AA AgsI
Table 1.29. Enzymes recognizing 5 bp long targets.
223
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.30. Enzymes recognizing 6 bp long targets.
Alphabetic List of Enzymes Recognizing 6 bp Long Targets
(continued on next page)
Specificity 5’→3’
FastDigest® enzyme
Conventional enzyme
AA↓CGTT FastDigest® AclI (Psp1406I) Psp1406I (AclI)A↓AGCTT FastDigest® HindIII HindIII↓(8/13)AAG(N)5CTT(13/8)↓ FalIAAT↓ATT FastDigest® SspI SspIA↓CATGT PscI (PciI)A↓CCGGT FastDigest® AgeI (BshTI) BshTI (AgeI)ACCTGC(4/8)↓* FastDigest® BspMI (BveI) BveI (BspMI)A↓CGCGT FastDigest® MluI MluI↓(9/12)AC(N)5CTCC(10/7)↓* BsaXIA↓CRYGT AflIIIA↓CTAGT FastDigest® SpeI (BcuI) BcuI (SpeI)ACTGGG(5/4)↓* BfiI (BmrI)A↓GATCT FastDigest® BglII BglIIAGC↓GCT FastDigest® AfeI (Eco47III) Eco47III (AfeI)AGG↓CCT FastDigest® StuI (Eco147I) Eco147I (StuI)AGT↓ACT FastDigest® ScaI ScaIAT↓CGAT FastDigest® ClaI (Bsu15I) Bsu15I (ClaI)ATGCA↓T FastDigest® NsiI (Mph1103I) Mph1103I (NsiI)AT↓TAAT FastDigest® AseI (VspI) VspI (AseI)C↓AATTG FastDigest® MfeI (MunI) MunI (MfeI)CACCCA(11/9)↓* TaqIICACGAG(-5/-1)↓* BauI (BssSI)CACGTC(-3/-3)↓* AjiI (BmgBI)CAC↓GTG FastDigest® PmlI (Eco72I) Eco72I (PmlI)CACNNN↓GTG FastDigest® DraIII (AdeI) AdeI (DraIII)CACNN↓NNGTG FastDigest® AleI (OliI) OliI (AleI)↓(8/13)CAC(N)6TCC(12/7)↓* TstI↓(0/2)CACTGC* BtsICAGCAG(25/27)↓* EcoP15ICAG↓CTG FastDigest® PvuII PvuIICAGNNN↓CTG FastDigest® AlwNI (CaiI) CaiI (AlwNI)CA↓TATG FastDigest® NdeI NdeI↓(14/10)CATCGC* BtgZI↓(0/2)CATTGC* FastDigest® BsrDI (BseMI) BseMI (BsrDI)CAYNN↓NNRTG FastDigest® MslI (RseI) RseI (MslI)CCANNNNN↓NNNNTGG XcmICCANNNNN↓NTGG FastDigest® BstXI BstXICCANNNN↓NTGG FastDigest® PflMI (Van91I) Van91I (PflMI)C↓CATGG FastDigest® NcoI NcoICCCAGC(-5/-1)↓* BseYICCCAGC(-1/-5)↓* FastDigest® PspFI GsaI↓(4/5)CCCAGT* BfiI (BmrI)C↓CCGGG Cfr9I (XmaI)CCC↓GGG FastDigest® SmaI SmaICCGC↓GG Cfr42I (SacII)CCGCTC(-3/-3)↓* FastDigest® BsrBI (MbiI) MbiI (BsrBI)C↓CRYGG BtgIC↓CTAGG FastDigest® AvrII (XmaJI) XmaJI (AvrII)CCTNAGC(-5/-2)↓* FastDigest® Bpu10I Bpu10ICC↓TNAGG FastDigest® Bsu36I (Eco81I) Eco81I (Bsu36I)CCTNN↓NNNAGG FastDigest® EcoNI (XagI) XagI (EcoNI)C↓CWWGG FastDigest® StyI (Eco130I) Eco130I (StyI)↓(10/12)CGA(N)6TGC(12/10)↓* BcgICGAT↓CG FastDigest® PvuI PvuIC↓GGCCG FastDigest® EagI (Eco52I) Eco52I (EagI)CGRY↓CG FastDigest® BsiEI (Bsh1285I) Bsh1285I (BsiEI)C↓GTACG FastDigest® BsiWI (Pfl23II) Pfl23II (BsiWI)CGTCTC(1/5)↓* FastDigest® BsmBI (Esp3I) Esp3I (BsmBI)CMG↓CKG MspA1I↓(14/16)CTCAAG* BpuEI↓(14/16)CTCCAG* FastDigest® BpmI (GsuI) GsuI (BpmI)↓(8/10)CTCCTC* BseRIC↓TCGAG FastDigest® XhoI XhoI↓(19/21)CTCGGC* NmeAIII
Specificity 5’→3’
FastDigest® enzyme
Conventional enzyme
CTCGTG(-5/-1)↓* BauI (BssSI)CTCTTC(1/4)↓* FastDigest® EarI (Eam1104I) Eam1104I (EarI)CTGAAG(16/14)↓* FastDigest® AcuI (Eco57I) Eco57I (AcuI)↓(14/16)CTGCAC* BsgICTGCA↓G FastDigest® PstI PstI↓(27/25)CTGCTG* EcoP15ICTGGAG(16/14)↓* FastDigest® BpmI (GsuI) GsuI (BpmI)CTGRAG(16/14)↓* Eco57MIC↓TRYAG FastDigest® SfcI (BfmI) BfmI (SfcI)C↓TTAAG FastDigest® AflII (BspTI) BspTI (AflII)↓(14/16)CTTCAG* FastDigest® AcuI (Eco57I) Eco57I (AcuI)CTTGAG(16/14)↓* BpuEI↓(14/16)CTYCAG* Eco57MIC↓TYRAG SmoI (SmlI)C↓YCGRG FastDigest® AvaI (Eco88I) Eco88I (AvaI)CY↓CGRG BmeT110IGAAGAC(2/6)↓* FastDigest® BbsI (BpiI) BpiI (BbsI)↓(4/1)GAAGAG* FastDigest® EarI (Eam1104I) Eam1104I (EarI)GAANN↓NNTTC FastDigest® PdmI PdmI (XmnI)GAATGC(1/-1)↓* FastDigest® Mva1269I Mva1269I (BsmI)G↓AATTC FastDigest® EcoRI EcoRI↓(8/13-14)GAB(N)5RTC(13-14/8)↓* Hin4IGACCGA(11/9)↓* TaqIIGAC↓GTC ZraIGACGT↓C FastDigest® AatII AatIIGACGTG(-3/-3)↓* AjiI (BmgBI)GACN↓NNGTC FastDigest® PsyI PsyI (Tth111I)GACNN↓NNGTC FastDigest® PshAI (BoxI) BoxI (PshAI)GACNNN↓NNGTC FastDigest® Eam1105I Eam1105I (AhdI)GACNNNN↓NNGTC FastDigest® DrdI (AasI) AasI (DrdI)↓(5/1)GAGACC* FastDigest® Eco31I Eco31I (BsaI)↓(5/1)GAGACG* FastDigest® BsmBI (Esp3I) Esp3I (BsmBI)GAGCGG(-3/-3)↓* FastDigest® BsrBI (MbiI) MbiI (BsrBI)GAGCT↓C FastDigest® SacI SacIGAG↓CTC FastDigest® Ecl136II Ecl136II (EcoICRI)GAGGAG(10/8)↓* BseRI↓(8/13)GAG(N)5CTC(13/8)↓ FastDigest® BplI BplIGAT↓ATC FastDigest® EcoRV (Eco32I) Eco32I (EcoRV)GATNN↓NNATC FastDigest® BsaBI (BseJI) BseJI (BsaBI)(8/13-14)GAY(N)5VTC(13-14/8)↓* Hin4IGCAATG(2/0)↓* FastDigest® BsrDI (BseMI) BseMI (BsrDI)↓(8/4)GCAGGT* FastDigest® BspMI (BveI) BveI (BspMI)GCAGTG(2/0)↓* BtsI↓(10/12)GCA(N)6TCG(12/10)↓* BcgI↓(10/12)GCA(N)6TGC(12/10)↓ AlfIGCANNNN↓NTGC BstAPIGCATG↓C FastDigest® SphI (PaeI) PaeI (SphI)↓(-1/1)GCATTC* FastDigest® Mva1269I Mva1269I (BsmI)GCCGAG(21/19)↓* NmeAIIIG↓CCGGC NgoMIVGCC↓GGC FastDigest® NaeI (PdiI) PdiI (NaeI)GCCNNNN↓NGGC FastDigest® BglI BglIGCGATG(10/14)↓* BtgZIG↓CGCGC FastDigest® BssHII (PfeI) PauI (BssHII)GCTAG↓C FastDigest® BmtI (BspOI) BspOI (BmtI)G↓CTAGC FastDigest® NheI NheIGCTGGG(-5/-1)↓* BseYIGCTGGG(-1/-5)↓* FastDigest® PspFI GsaIGC↓TNAGC FastDigest® BlpI (Bpu1102I) Bpu1102I (BlpI)GCTNAGG(-5/-2)↓* FastDigest® Bpu10I Bpu10IGDGCH↓C FastDigest® Bsp1286I (SduI) SduI (Bsp1286I)↓(7/10)GGAG(N)5GT(12/9)↓* BsaXI↓(7/12)GGA(N)6GTG(13/8)↓* TstI↓(5/6)GGATAC* BfuI (BciVI)
224
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.30. Enzymes recognizing 6 bp long targets.
Note* Asymetric sequences.• Fermentas FastDigest® enzymes are shown in ruby.• Fermentas conventional enzymes are shown in orange.
Single letter codeR = G or A; K = G or T; B = C, G or T; M = A or C;Y = C or T; S = C or G; D = A, G or T; V = A, C or G.W = A or T; H = A, C or T; N = G, A, T or C;
Specificity 5’→3’
FastDigest® enzyme
Conventional enzyme
G↓GATCC FastDigest® BamHI BamHIGGC↓GCC FastDigest® EheI EheI (SfoI)GG↓CGCC NarIG↓GCGCC SspDI (KasI)GGCGC↓C BbeIGGCGGA(11/9)↓* EciIG↓GGCCC FastDigest® Bsp120I Bsp120I (PspOMI)GGGCC↓C FastDigest® ApaI ApaIGGTAC↓C FastDigest® KpnI KpnIG↓GTACC FastDigest® Acc65I Acc65I (Asp718I)GGTCTC(1/5)↓* FastDigest® Eco31I Eco31I (BsaI)G↓GTNACC FastDigest® Eco91I Eco91I (BstEII)G↓GYRCC FastDigest® BanI (BshNI) BshNI (BanI)GKGCM↓C FastDigest® Bme1580I (BseSI) BseSI (Bme1580I)GR↓CGYC FastDigest® BsaHI (Hin1I) Hin1I (BsaHI)GRGCY↓C Eco24I (BanII)GTA↓TAC FastDigest® BstZ17I (Bst1107I) Bst1107I (BstZ17I)GTATCC(6/5)↓* BfuI (BciVI)G↓TCGAC FastDigest® SalI SalI↓(6/2)GTCTTC* FastDigest® BbsI (BpiI) BpiI (BbsI)G↓TGCAC FastDigest® ApaLI (Alw44I) Alw44I (ApaLI)GTGCAG(16/14)↓* BsgIGT↓MKAC FastDigest® AccI (XmiI) XmiI (AccI)GTT↓AAC FastDigest® HpaI (KspAI) KspAI (HpaI)↓(18/20)GTYGGA* MmeIGTY↓RAC FastDigest® HincII HincII (HindII)GWGCW↓C FastDigest® Alw21I Alw21I (BsiHKAI)R↓AATTY FastDigest® XapI XapI (ApoI)RCATG↓Y FastDigest® NspI (XceI) XceI (NspI)R↓CCGGY FastDigest® BsrFI (Cfr10I) Cfr10I (BsrFI)R↓GATCY FastDigest® PsuI PsuI (BstYI)RGCGC↓Y FastDigest® HaeII (BfoI) HaeIIRG↓GNCCY FastDigest® EcoO109I EcoO109I (DraII)TAC↓GTA FastDigest® SnaBI (Eco105I) Eco105I (SnaBI)TARCCA(11/9)↓* TsoIT↓CATGA FastDigest® BspHI (PagI) PagI (BspHI)↓(9/11)TCCGCC* EciIT↓CCGGA FastDigest® Kpn2I Kpn2I (BspEI)T↓CCNGGA FastDigest® PfoI PfoITCCRAC(20/18)↓* MmeITCG↓CGA FastDigest® NruI (RruI) Bsp68I (NruI)↓(9/11)TCGGTC* TaqIIT↓CTAGA FastDigest® XbaI XbaI↓(10/12)TGA(N)6TCA(12/10)↓ BdaIT↓GATCA FastDigest® BclI BclITGC↓GCA FastDigest® FspI (NsbI) NsbI (FspI)TGG↓CCA FastDigest® MscI (MlsI) MlsI (MscI)↓(9/11)TGGGTG* TaqII↓(9/11)TGGYTA* TsoIT↓GTACA FastDigest® Bsp1407I Bsp1407I (BsrGI)TTA↓TAA FastDigest® PsiI (AanI) AanI (PsiI)TT↓CGAA FastDigest® Bsp119I Bsp119I (BstBI)TTT↓AAA FastDigest® DraI DraIW↓CCGGW BsaWIW↓GTACW FastDigest® TatI TatIYAC↓GTR FastDigest® BsaAI (Ppu21I) Ppu21I (BsaAI)Y↓GGCCR CfrI (EaeI)
Alphabetic List of Enzymes Recognizing 8 bp Long Targets
Alphabetic List of Enzymes Recognizing 7 bp Long Targets
Table 1.31. Enzymes recognizing 7 bp long targets.
Specificity 5’→3’
FastDigest® enzyme
Conventional enzyme
A↓CCWGGT FastDigest® SexAI (CsiI) SexAI↓(10/15)AC(N)4GTAYC(12/7)↓* BaeI↓(11/13)CAA(N)5GTGG(12/10)↓* CspCICACCTGC(4/8)↓* AarI↓(6/11)CCAA(N)7TTC(12/7)↓* FastDigest® AjuI AjuI↓(10/12)CCAC(N)5TTG(13/11)↓* CspCICC↓CWGGG PasICCTCAGC(-5/-2)↓* BbvCICG↓GWCCG FastDigest® RsrII (CpoI) CpoI (RsrII)↓(6/11)CRAA(N)6GTC(13/8)↓* ArsI↓(7/12)GAAC(N)5CTC(13/8)↓* PpiI↓(7/12)GAAC(N)6TAC(12/7)↓* PsrI↓(7/12-13)GAAC(N)6TCC(12-13/7)↓* AloI↓(4/1)GAAGAGC* FastDigest® SapI (LguI) LguI (SapI)↓(7/12)GAAG(N)6TAC(12/7)↓* BarI↓(7/12)GAA(N)7TTGG(11/6)↓* FastDigest® AjuI AjuI↓(8/13)GAC(N)6TTYG(11/6)↓* ArsI↓(8/13)GAG(N)5GTTC(12/7)↓* PpiI↓(8/4)GCAGGTG* AarIGCTCTTC(1/4)↓* FastDigest® SapI (LguI) LguI (SapI)GCTGAGG(-5/-2)↓* BbvCI↓(7/12-13)GGA(N)6GTTC(12-13/7)↓* AloIGG↓GWCCC FastDigest® SanDI (KflI) SanDI↓(7/12)GRTACNNNNGT(15/10)↓* BaeI↓(7/12)GTA(N)6CTTC(12/7)↓* BarI↓(7/12)GTA(N)6GTTC(12/7)↓* PsrIRG↓GWCCY FastDigest® PpuMI (Psp5II) Psp5II (PpuMI)
Specificity 5’→3’FastDigest® enzyme
Conventional enzyme
ATTT↓AAAT FastDigest® SwaI (SmiI) SmiI (SwaI)CC↓TCGAGG AbsICCTGCA↓GG FastDigest® SbfI (SdaI) SdaI (SbfI)CG↓CCGGCG FastDigest® MreI MreI (Sse232I)CG↓CGCGCG FastDigest® MauBI MauBICG↓TCGACG SgrDICR↓CCGGYG SgrAIGCCC↓GGGC SrfIGCGAT↓CGC FastDigest® AsiSI (SfaAI) SfaAI (AsiSI)GC↓GGCCGC FastDigest® NotI NotIGGCCGG↓CC FseIGGCCNNNN↓NGGCC FastDigest® SfiI SfiIGG↓CGCGCC FastDigest® AscI (SgsI) SgsI (AscI)GTTT↓AAAC FastDigest® MssI MssI (PmeI)RTGC↓GCAY FastDigest® FspAI FspAITTAAT↓TAA FastDigest® PacI PacIVC↓TCGAGB PspXI
Table 1.32. Enzymes recognizing 8 bp long targets.
225
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.33. Commercial restriction enzymes generating 5’-protruding ends.
Commercial Restriction Enzymes Generating 5’-protruding Ends
(continued on next page)
Recognition sequence
FastDigest® enzyme
Conventional enzyme
5’-protruding end (5’→ 3’)1 nt 2 nt 3 nt 4 nt 5 nt
AA↓CGTT FastDigest® AclI (Psp1406I) Psp1406I (AclI) CGA↓AGCTT FastDigest® HindIII HindIII AGCT↓AATT FastDigest® Tsp509I (TasI) TasI (Tsp509I) AATTA↓CATGT PscI (PciI) CATGA↓CCGGT FastDigest® AgeI (BshTI) BshTI (AgeI) CCGGACCTGC(4/8)↓* FastDigest® BspMI (BveI) BveI (BspMI) NNNNA↓CCWGGT FastDigest® SexAI (CsiI) SexAI CCWGGA↓CGCGT FastDigest® MluI MluI CGCGACGGC(12/14)↓* BceAI NNA↓CGT MaeII CGA↓CRYGT AflIII CRYGA↓CTAGT FastDigest® SpeI (BcuI) BcuI (SpeI) CTAGA↓GATCT FastDigest® BglII BglII GATCAT↓CGAT FastDigest® ClaI (Bsu15I) Bsu15I (ClaI) CGAT↓TAAT FastDigest® AseI (VspI) VspI (AseI) TAC↓AATTG FastDigest® MfeI (MunI) MunI (MfeI) AATTCACCTGC(4/8)↓* AarI NNNNCACGAG(-5/-1)↓* BauI (BssSI) ACGACAGCAG(25/27)↓* EcoP15I NNCA↓TATG FastDigest® NdeI NdeI TA↓(13/9)CATCC* FastDigest® FokI FokI NNNN↓(14/10)CATCGC* BtgZI NNNN↓CATG FatI CATGC↓ATG CviAII ATCCATC(4/5)↓* BccI NC↓CATGG FastDigest® NcoI NcoI CATGCCCAGC(-5/-1)↓* BseYI CCAGCCCGC(4/6)↓* SmuI (FauI) NNC↓CCGGG Cfr9I (XmaI) CCGGCC↓CWGGG PasI CWGCCGC(-3/-1)↓* FastDigest® AciI (SsiI) SsiI (AciI) CG
C↓CGGFastDigest® HpaIIFastDigest® MspI
HpaIIMspI (HpaII)
CG
↓CCNGG StyD4I CCNGGCC↓NGG FastDigest® ScrFI (Bme1390I) Bme1390I (ScrFI) NC↓CNNGG FastDigest® BsaJI (BseDI) BseDI (BsaJI) CNNGC↓CRYGG BtgI CRYGCC↓SGG FastDigest® NciI (BcnI) BcnI (NciI) SC↓CTAGG FastDigest® AvrII (XmaJI) XmaJI (AvrII) CTAGCCTCAGC(-5/-2)↓* BbvCI TCACC↓TCGAGG AbsI TCGACCTNAGC(-5/-2)↓* FastDigest® Bpu10I Bpu10I TNACC↓TNAGG FastDigest® Bsu36I (Eco81I) Eco81I (Bsu36I) TNACCTNN↓NNNAGG FastDigest® EcoNI (XagI) XagI (EcoNI) N↓CCWGG EcoRII CCWGGCC↓WGG FastDigest® MvaI MvaI (BstNI) WC↓CWWGG FastDigest® StyI (Eco130I) Eco130I (StyI) CWWGCG↓CCGGCG FastDigest® MreI MreI (Sse232I) CCGGCG↓CGCGCG FastDigest® MauBI MauBI CGCGC↓GGCCG FastDigest® EagI (Eco52I) Eco52I (EagI) GGCCCG↓GWCCG FastDigest® RsrII (CpoI) CpoI (RsrII) GWCC↓GTACG FastDigest® BsiWI (Pfl23II) Pfl23II (BsiWI) GTAC5mCNNG(9/13)↓ SgeI NNNNCG↓TCGACG SgrDI TCGACGTCTC(1/5)↓* FastDigest® BsmBI (Esp3I) Esp3I (BsmBI) NNNNCR↓CCGGYG SgrAI CCGG
226
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
(continued on next page)
Table 1.33. Commercial restriction enzymes generating 5’-protruding ends.
Recognition sequence
FastDigest® enzyme
Conventional enzyme
5’-protruding end (5’→ 3’)1 nt 2 nt 3 nt 4 nt 5 nt
C↓TAG FastDigest® BfaI (FspBI) FspBI (BfaI) TAC↓TCGAG FastDigest® XhoI XhoI TCGACTCGTG(-5/-1)↓* BauI (BssSI) TCGTCTCTTC(1/4)↓* FastDigest® EarI (Eam1104I) Eam1104I (EarI) NNN↓(27/25)CTGCTG* EcoP15I NNC↓TNAG FastDigest® DdeI (HpyF3I) HpyF3I (DdeI) TNAC↓TRYAG FastDigest® SfcI (BfmI) BfmI (SfcI) TRYAC↓TTAAG FastDigest® AflII (BspTI) BspTI (AflII) TTAAC↓TYRAG SmoI (SmlI) TYRAC↓YCGRG FastDigest® AvaI (Eco88I) Eco88I (AvaI) YCGRCY↓CGRG BmeT110I CGGAAGAC(2/6)↓* FastDigest® BbsI (BpiI) BpiI (BbsI) NNNN↓(4/1)GAAGAG* FastDigest® EarI (Eam1104I) Eam1104I (EarI) NNN↓(4/1)GAAGAGC* FastDigest® SapI (LguI) LguI (SapI) NNNG↓AATTC FastDigest® EcoRI EcoRI AATTGACGC(5/10)↓* FastDigest® HgaI (CseI) CseI (HgaI) NNNNNGACN↓NNGTC FastDigest® PsyI PsyI (Tth111I) N↓(5/4)GACTC* PleI N↓(5/1)GAGAC* FastDigest® Alw26I Alw26I (BsmAI) NNNN↓(5/1)GAGACC* FastDigest® Eco31I Eco31I (BsaI) NNNN↓(5/1)GAGACG* FastDigest® BsmBI (Esp3I) Esp3I (BsmBI) NNNNGAGTC(4/5)↓* PleI NG↓ANTC FastDigest® HinfI HinfI ANT
↓GATCFastDigest® MboIFastDigest® Sau3AI (Bsp143I)
MboIBsp143I (Sau3AI)
GATC
↓(5/4)GATCC* BspPI (AlwI) N↓(9/5)GATGC* FastDigest® SfaNI (BmsI) LweI (SfaNI) NNNN↓(5/4)GATGG* BccI NG↓AWTC FastDigest® TfiI (PfeI) PfeI (TfiI) AWT
GCAGC(8/12)↓*FastDigest® BbvI (Lsp1109I)FastDigest® BseXI (BbvI)
Lsp1109I (BbvI)BseXI (BbvI)
NNNN
↓(8/4)GCAGGT* FastDigest® BspMI (BveI) BveI (BspMI) NNNN↓(8/4)GCAGGTG* AarI NNNNGCATC(5/9)↓* FastDigest® SfaNI (BmsI) LweI (SfaNI) NNNNG↓CCGGC NgoMIV CCGG↓(14/12)GCCGT* BceAI NNGCGATG(10/14)↓* BtgZI NNNNG↓CGC FastDigest® HinP1I (Hin6I) Hin6I (HinP1I) CGG↓CGCGC FastDigest® BssHII (PteI) PauI (BssHII) CGCGGCGG(-3/-1)↓* FastDigest® AciI (SsiI) SsiI (AciI) CGGC↓GGCCGC FastDigest® NotI NotI GGCC↓(6/4)GCGGG* SmuI (FauI) NN↓(10/5)GCGTC* FastDigest® HgaI (CseI) CseI (HgaI) NNNNNGC↓NGC FastDigest® Fnu4HI (SatI) SatI (Fnu4HI) NG↓CTAGC FastDigest® NheI NheI CTAGGCTCTTC(1/4)↓* FastDigest® SapI (LguI) LguI (SapI) NNNGCTGAGG(-5/-2)↓* BbvCI TGA
↓(12/8)GCTGC*FastDigest® BbvI (Lsp1109I)FastDigest® BseXI (BbvI)
Lsp1109I (BbvI)BseXI (BbvI)
NNNN
GCTGGG(-5/-1)↓* BseYI CTGGGC↓TNAGC FastDigest® BlpI (Bpu1102I) Bpu1102I (BlpI) TNAGCTNAGG(-5/-2)↓* FastDigest® Bpu10I Bpu10I TNAG↓CWGC TseI CWGGGATC(4/5)↓* BspPI (AlwI) NG↓GATCC FastDigest® BamHI BamHI GATCGGATG(9/13)↓* FastDigest® FokI FokI NNNNGG↓CGCC NarI CGG↓GCGCC SspDI (KasI) GCGCGG↓CGCGCC FastDigest® AscI (SgsI) SgsI (AscI) CGCGGGGAC(10/14)↓* FastDigest® BsmFI (FaqI) FaqI (BsmFI) NNNN
227
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.33. Commercial restriction enzymes generating 5’-protruding ends.
Recognition sequence
FastDigest® enzyme
Conventional enzyme
5’-protruding end (5’→ 3’)1 nt 2 nt 3 nt 4 nt 5 nt
G↓GGCCC FastDigest® Bsp120I Bsp120I (PspOMI) GGCCGG↓GWCCC FastDigest® SanDI (KflI) SanDI GWCG↓GNCC FastDigest® Sau96I (Cfr13I) Cfr13I (Sau96I) GNCGG↓NCC BmgT120I NG↓GTACC FastDigest® Acc65I Acc65I (Asp718I) GTACGGTCTC(1/5)↓* FastDigest® Eco31I Eco31I (BsaI) NNNNG↓GTNACC FastDigest® Eco91I Eco91I (BstEII) GTNACG↓GWCC FastDigest® AvaII (Eco47I) Eco47I (AvaII) GWCG↓GYRCC FastDigest® BanI (BshNI) BshNI (BanI) GYRCGR↓CGYC FastDigest® BsaHI (Hin1I) Hin1I (BsaHI) CGG↓TAC FastDigest® Csp6I Csp6I (CviQI) TA↓(14/10)GTCCC* FastDigest® BsmFI (FaqI) FaqI (BsmFI) NNNNG↓TCGAC FastDigest® SalI SalI TCGAGTCTC(1/5)↓* FastDigest® Alw26I Alw26I (BsmAI) NNNN↓(6/2)GTCTTC* FastDigest® BbsI (BpiI) BpiI (BbsI) NNNNG↓TGCAC FastDigest® ApaLI (Alw44I) Alw44I (ApaLI) TGCAGT↓MKAC FastDigest® AccI (XmiI) XmiI (AccI) MK↓GTNAC MaeIII GTNAC↓GTSAC FastDigest® NmuCI NmuCI (Tsp45I) GTSACR↓AATTY FastDigest® XapI XapI (ApoI) AATTR↓CCGGY FastDigest® BsrFI (Cfr10I) Cfr10I (BsrFI) CCGGR↓GATCY FastDigest® PsuI PsuI (BstYI) GATCRG↓GNCCY FastDigest® EcoO109I EcoO109I (DraII) GNCRG↓GWCCY FastDigest® PpuMI (Psp5II) Psp5II (PpuMI) GWCT↓CATGA FastDigest® BspHI (PagI) PagI (BspHI) CATGT↓CCGGA FastDigest® Kpn2I Kpn2I (BspEI) CCGGT↓CCNGGA FastDigest® PfoI PfoI CCNGGT↓CGA FastDigest® TaqI TaqI CGTC↓NNGA Hpy188III NNT↓CTAGA FastDigest® XbaI XbaI CTAGT↓GATCA FastDigest® BclI BclI GATCT↓GTACA FastDigest® Bsp1407I Bsp1407I (BsrGI) GTAC
T↓TAAFastDigest® MseI (SaqAI)FastDigest® Tru1I
Tru1I (MseI) TA
TT↓CGAA FastDigest® Bsp119I Bsp119I (BstBI) CGVC↓TCGAGB PspXI TCGAW↓CCGGW BsaWI CCGGW↓GTACW FastDigest® TatI TatI GTACY↓GGCCR CfrI (EaeI) GGCC
Note* Asymetric sequences.• Fermentas FastDigest® enzymes are shown in ruby.• Fermentas conventional enzymes are shown in orange.
Single letter codeR = G or A; K = G or T; B = C, G or T;Y = C or T; S = C or G; D = A, G or T;W = A or T; H = A, C or T; N = G, A, T or C.M = A or C; V = A, C or G;
228
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
(continued on next page)
Table 1.34. Commercial restriction enzymes generating 3’-protruding ends.
Commercial Restriction Enzymes Generating 3’-protruding Ends
Recognition sequenceFastDigest® enzyme
Conventional enzyme
3’-protruding end (5’→ 3’)1 nt 2 nt 3 nt 4 nt 5 nt 9 nt
ACGGA(11/9)↓* TspGWI NNACGT↓ FastDigest® TaiI TaiI (MaeII) ACGTACN↓GT FastDigest® TaaI TaaI (HpyCH4III) NACTGG(1/-1)↓* FastDigest® BseNI BseNI (BsrI) GNACTGGG(5/4)↓* BfiI (BmrI) NASST↓ SetI ASSTATGAA(11/9)↓* TspDTI NNATGCA↓T FastDigest® NsiI (Mph1103I) Mph1103I (NsiI) TGCACACCCA(11/9)↓* TaqII NNCACNNN↓GTG FastDigest® DraIII (AdeI) AdeI (DraIII) NNN↓(0/2)CACTGC* BtsI NNCAGNNN↓CTG FastDigest® AlwNI (CaiI) CaiI (AlwNI) NNNCASTG(2/-7)↓ FastDigest® TspRI (TscAI) TscAI (TspRI) NNCASTGNN↓(0/2)CATCC* FastDigest® BseGI BseGI (BtsCI) NNCATG↓ FastDigest® NlaIII (Hin1II) Hin1II (NlaIII) CATG↓(0/2)CATTGC* FastDigest® BsrDI (BseMI) BseMI (BsrDI) NN↓(-1/1)CCAGT* FastDigest® BseNI BseNI (BsrI) NCCCANNNNN↓NNNNTGG XcmI NCCANNNNN↓NTGG FastDigest® BstXI BstXI NNNNCCANNNN↓NTGG FastDigest® PflMI (Van91I) Van91I (PflMI) NNNCCCAGC(-1/-5)↓* FastDigest® PspFI GsaI CCAG↓(4/5)CCCAGT* BfiI (BmrI) NCCGC↓GG Cfr42I (SacII) GCCCNNNNN↓NNGG FastDigest® BslI (BseLI) BseLI (BslI) NNNCCTC(7/6)↓* FastDigest® MnlI MnlI NCCTGCA↓GG FastDigest® SbfI (SdaI) SdaI (SbfI) TGCACCTTC(6/5)↓* HpyAV NCGAT↓CG FastDigest® PvuI PvuI ATCGRY↓CG FastDigest® BsiEI (Bsh1285I) Bsh1285I (BsiEI) RYCGWCG↓ Hpy99I CGWCG↓(14/16)CTCAAG* BpuEI NNCTCAG(9/7)↓* BspCNI NNCTCAG(10/8)↓* FastDigest® BspCNI (BseMII) BseMII (BspCNI) NN↓(14/16)CTCCAG* FastDigest® BpmI (GsuI) GsuI (BpmI) NN↓(8/10)CTCCTC* BseRI NN↓(19/21)CTCGGC* NmeAIII NNCTGAAG(16/14)↓* FastDigest® AcuI (Eco57I) Eco57I (AcuI) NN↓(7/9)CTGAG* BspCNI NN↓(8/10)CTGAG* FastDigest® BspCNI (BseMII) BseMII (BspCNI) NN↓(14/16)CTGCAC* BsgI NNCTGCA↓G FastDigest® PstI PstI TGCACTGGAG(16/14)↓* FastDigest® BpmI (GsuI) GsuI (BpmI) NNCTGRAG(16/14)↓* Eco57MI NN↓(14/16)CTTCAG* FastDigest® AcuI (Eco57I) Eco57I (AcuI) NNCTTGAG(16/14)↓* BpuEI NN↓(14/16)CTYCAG* Eco57MI NNGAAGA(8/7)↓* FastDigest® MboII MboII N↓(5/6)GAAGG* HpyAV NGAATGC(1/-1)↓* FastDigest® Mva1269I Mva1269I (BsmI) CNGACCGA(11/9)↓* TaqII NNGACGT↓C FastDigest® AatII AatII ACGTGACNNN↓NNGTC FastDigest® Eam1105I Eam1105I (AhdI) NGACNNNN↓NNGTC FastDigest® DrdI (AasI) AasI (DrdI) NNGAGCT↓C FastDigest® SacI SacI AGCT↓(6/7)GAGG* FastDigest® MnlI MnlI NGAGGAG(10/8)↓* BseRI NNGAT↓C BstKTI ATGCAATG(2/0)↓* FastDigest® BsrDI (BseMI) BseMI (BsrDI) NNGCAGTG(2/0)↓* BtsI NN
229
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Table 1.34. Commercial restriction enzymes generating 3’-protruding ends.
Note* Asymetric sequences.• Fermentas FastDigest® enzymes are shown in ruby.• Fermentas conventional enzymes are shown in orange.
Single letter codeR = G or A; K = G or T; B = C, G or T;Y = C or T; S = C or G; D = A, G or T;W = A or T; H = A, C or T; N = G, A, T or C.M = A or C; V = A, C or G;
Recognition sequenceFastDigest® enzyme
Conventional enzyme
3’-protruding end (5’→ 3’)1 nt 2 nt 3 nt 4 nt 5 nt 9 nt
GCANNNN↓NTGC BstAPI NNNGCATG↓C FastDigest® SphI (PaeI) PaeI (SphI) CATG↓(-1/1)GCATTC* FastDigest® Mva1269I Mva1269I (BsmI) NGGCCGAG(21/19)↓* NmeAIII NNGCCNNNN↓NGGC FastDigest® BglI BglI NNNGCGAT↓CGC FastDigest® AsiSI (SfaAI) SfaAI (AsiSI) ATGCG↓C FastDigest® HhaI HhaI CGGCNNNNN↓NNGC FastDigest® HpyF10VI HpyF10VI (MwoI) NNNGCSG↓C FastDigest® TauI TauI CSGGCTAG↓C FastDigest® BmtI (BspOI) BspOI (BmtI) CTAGGCTGGG(-1/-5)↓* FastDigest® PspFI GsaI CTGGGDGCH↓C FastDigest® Bsp1286I (SduI) SduI (Bsp1286I) DGCH↓(5/6)GGATAC* BfuI (BciVI) NGGATG(2/0)↓* FastDigest® BseGI BseGI (BtsCI) NNGGCCGG↓CC FseI CCGGGGCCNNNN↓NGGCC FastDigest® SfiI SfiI NNNGGCGC↓C BbeI GCGCGGCGGA(11/9)↓* EciI NNGGGCC↓C FastDigest® ApaI ApaI GGCCGGTAC↓C FastDigest® KpnI KpnI GTACGGTGA(8/7)↓* HphI N
GKGCM↓CFastDigest® Bme1580I (BseSI)
BseSI (Bme1580I) KGCM
GRGCY↓C Eco24I (BanII) RGCYGTATCC(6/5)↓* BfuI (BciVI) NGTGCAG(16/14)↓* BsgI NN↓(18/20)GTYGGA* MmeI NNGWGCW↓C FastDigest® Alw21I Alw21I (BsiHKAI) WGCWRCATG↓Y FastDigest® NspI (XceI) XceI (NspI) CATGRGCGC↓Y FastDigest® HaeII (BfoI) HaeII GCGCTARCCA(11/9)↓* TsoI NN↓(7/8)TCACC* HphI N↓(9/11)TCCGCC* EciI NN↓(9/11)TCCGT* TspGWI NNTCCRAC(20/18)↓* MmeI NN↓(9/11)TCGGTC* TaqII NNTCN↓GA Hpy188I N↓(7/8)TCTTC* FastDigest® MboII MboII N↓(9/11)TGGGTG* TaqII NN↓(9/11)TGGYTA* TsoI NNTTAAT↓TAA FastDigest® PacI PacI AT↓(9/11)TTCAT* TspDTI NNTTS↓AA AgsI S
230
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.35. Commercial restriction enzymes generating blunt ends.
Commercial Restriction Enzymes Generating Blunt Ends
Table 1.36. Commercial restriction enzymes cleaving DNA on the both sides of their recognition sequence.
Commercial Restriction Enzymes Cleaving DNA on the Both Sides of their Recognition Sequence
Reccognition sequence
FastDigest®
enzymeConventional enzyme
AAT↓ATT FastDigest® SspI SspIAGC↓GCT FastDigest® AfeI (Eco47III) Eco47III (AfeI)AG↓CT FastDigest® AluI AluIAGG↓CCT FastDigest® StuI (Eco147I) Eco147I (StuI)AGT↓ACT FastDigest® ScaI ScaIATTT↓AAAT FastDigest® SwaI (SmiI) SmiI (SwaI)CACGTC(-3/-3)↓* AjiI (BmgBI)CAC↓GTG FastDigest® PmlI (Eco72I) Eco72I (PmlI)CACNN↓NNGTG FastDigest® AleI (OliI) OliI (AleI)CAG↓CTG FastDigest® PvuII PvuIICAYNN↓NNRTG FastDigest® MslI (RseI) RseI (MslI)CCC↓GGG FastDigest® SmaI SmaICCGCTC(-3/-3)↓* FastDigest® BsrBI (MbiI) MbiI (BsrBI)CG↓CG FastDigest® Bsh1236I Bsh1236I (BstUI)CMG↓CKG MspA1IGAANN↓NNTTC FastDigest® PdmI PdmI (XmnI)GAC↓GTC ZraIGACGTG(-3/-3)↓* AjiI (BmgBI)GACNN↓NNGTC FastDigest® PshAI (BoxI) BoxI (PshAI)↓(5/5)GACTC* FastDigest® MlyI (SchI) SchI (MlyI)GAGCGG(-3/-3)↓* FastDigest® BsrBI (MbiI) MbiI (BsrBI)GAG↓CTC FastDigest® Ecl136II Ecl136II (EcoICRI)GAGTC(5/5)↓* FastDigest® MlyI (SchI) SchI (MlyI)GAT↓ATC FastDigest® EcoRV (Eco32I) Eco32I (EcoRV)GA↓TC FastDigest® DpnI DpnIGATNN↓NNATC FastDigest® BsaBI (BseJI) BseJI (BsaBI)GCCC↓GGGC SrfIGCC↓GGC FastDigest® NaeI (PdiI) PdiI (NaeI)GC↓GC GlaIGCN↓NGC Cac8IGG↓CC FastDigest® HaeIII (BsuRI) BsuRI (HaeIII)GGC↓GCC FastDigest® EheI EheI (SfoI)GGN↓NCC FastDigest® NlaIV (BspLI) BspLI (NlaIV)GT↓AC FastDigest® RsaI RsaIGTA↓TAC FastDigest® BstZ17I (Bst1107I) Bst1107I (BstZ17I)GTN↓NAC FastDigest® Hpy8I Hpy8I (MjaIV)GTT↓AAC FastDigest® HpaI (KspAI) KspAI (HpaI)GTTT↓AAAC FastDigest® MssI MssI (PmeI)GTY↓RAC FastDigest® HincII HincII (HindII)RG↓CY CviJIRTGC↓GCAY FastDigest® FspAI FspAITAC↓GTA FastDigest® SnaBI (Eco105I) Eco105I (SnaBI)TCG↓CGA FastDigest® NruI (RruI) Bsp68I (NruI)TG↓CA HpyCH4VTGC↓GCA FastDigest® FspI (NsbI) NsbI (FspI)TGG↓CCA FastDigest® MscI (MlsI) MlsI (MscI)TTA↓TAA FastDigest® PsiI (AanI) AanI (PsiI)TTT↓AAA FastDigest® DraI DraIYAC↓GTR FastDigest® BsaAI (Ppu21I) Ppu21I (BsaAI)YA↓TR FaiI
Note* Asymetric sequences.• Fermentas FastDigest® enzymes are shown in ruby.• Fermentas conventional enzymes are shown in orange.
Single letter codeR = G or A; K = G or T; B = C, G or T;Y = C or T; S = C or G; D = A, G or T;W = A or T; H = A, C or T; N = G, A, T or C.M = A or C; V = A, C or G;
Recognition sequenceFastDigest® enzyme
Conventional enzyme
↓(8/13)AAG(N)5CTT(13/8)↓ FalI↓(9/12)AC(N)5CTCC(10/7)↓ BsaXI↓(10/15)AC(N)4GTAYC(12/7)↓ BaeI↓(11/13)CAA(N)5GTGG(12/10)↓ CspCI↓(8/13)CAC(N)6TCC(12/7)↓ TstI↓(6/11)CCAA(N)7TTC(12/7)↓ FastDigest® AjuI AjuI↓(10/12)CCAC(N)5TTG(13/11)↓ CspCI↓(10/12)CGA(N)6TGC(12/10)↓ BcgI↓(6/11)CRAA(N)6GTC(13/8)↓ ArsI↓(7/12)GAA(N)7TTGG(11/6)↓ FastDigest® AjuI AjuI↓(7/12)GAAC(N)5CTC(13/8)↓ PpiI↓(7/12)GAAC(N)6TAC(12/7)↓ PsrI↓(7/12-13)GAAC(N)6TCC(12-13/7)↓ AloI↓(7/12)GAAG(N)6TAC(12/7)↓ BarI↓(8/13-14)GAB(N)5RTC(13-14/8)↓ Hin4I↓(8/13)GAC(N)6TTYG(11/6)↓ ArsI↓(8/13)GAG(N)5CTC(13/8)↓ FastDigest® BplI BplI↓(8/13)GAG(N)5GTTC(12/7)↓ PpiI↓(8/13-14)GAY(N)5VTC(13-14/8)↓ Hin4I↓(10/12)GCA(N)6TCG(12/10)↓ BcgI↓(10/12)GCA(N)6TGC(12/10)↓ AlfI↓(7/12)GGA(N)6GTG(13/8)↓ TstI↓(7/12-13)GGA(N)6GTTC(12-13/7)↓ AloI↓(7/10)GGAG(N)5GT(12/9)↓ BsaXI↓(7/12)GRTAC(N)4GT(15/10)↓ BaeI↓(7/12)GTA(N)6CTTC(12/7)↓ BarI↓(7/12)GTA(N)6GTTC(12/7)↓ PsrI↓(10/12)TGA(N)6TCA(12/10)↓ BdaI
231
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Troubleshooting Guide
3 Diffuse DNA bands on gel
3.1 Gel shift
3.2 Contaminated
reagents
1 Incomplete
digestion, no digestion
Assess enzyme activity with control DNA
1.1 Inactive enzyme Active
enzyme
1.3 Unsuitable or contaminated
DNA
1.4 Water
contains impurities
2 Unexpected
cleavage pattern
2.1 Star
activity
2.2 Contamination with another
RE
2.3 Contamination with another
DNA
2.4 Incomplete DNA
digestion
2.5 Gel shift
2.6 Unexpected
sites in template DNA
1.2.1 Suboptimal di-
gestion protocol
1.2 Suboptimal
reaction conditions
1.2.2 Improper enzyme dilution
1.2.3 Improper reaction
assembly
1.2.4 Excess glycerol
1.2.5 Suboptimal DNA
concentration
Evaluate inhibition by
template DNA solution
No inhibition
1.3.1 Contaminants
in DNA solution
1.3.2 Absence of recognition
sites
1.3.3 Cleavage
blocked by methylation
Digestion problem
1.3.4 Structure of
DNA substrate
1.3.4.4 Site
preference
1.3.4.3 At least two
sites required
1.3.4.1 Supercoiled
plasmid DNA
1.3.4.2 Proximity of site to DNA
ends
Inhibition
232
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Table 1.37. Troubleshooting guide for DNA digestion.
Problem Possible cause and recommended solution
1. Incomplete digestion or no digestion
Assess enzyme activityThe restriction enzyme may lose activity due to improper storage or handling. Perform a digestion reaction with 1 µg of a standard control DNA, e.g. Lambda DNA (dam–, dcm–) (#SD0021).
1.1. Inactive enzyme. If the enzyme does not cut the control DNA:• Check the expiration date.• Verify that the enzyme has been stored at -20°C.• Check the temperature of your freezer. Do not allow the temperature go below -20°C as the enzyme may
freeze and multiple freeze thaw cycles (more than 3 cycles) may result in reduced enzyme activity.
1.2. Suboptimal reaction conditions. 1.2.1. Suboptimal digestion protocol. • Follow digestion protocol specified for the restriction enzyme and type of substrate DNA. All FastDigest®
enzymes are experimentally tested on Lambda DNA (or other control substrate DNA), plasmid and genomic DNA as well as PCR products. Please refer to Table 1.3 on p.71 for specific recommendations.
• For FastDigest® enzymes use FastDigest® or FastDigest® Green Buffer. All FastDigest® enzymes are 100% active in these buffers.
• For conventional restriction enzymes use the recommended reaction buffer supplied with the enzyme. For double digestions follow the recommendations of the DoubleDigest™ engine at www.fermentas.com/doubledigest.
• Use additives where required.• Perform the reaction at the optimal temperature specified for the restriction enzyme. For double digestions
with enzymes requiring different incubation temperatures perform sequential DNA cleavage: complete the first digestion reaction at the lower temperature, add the second enzyme and increase the digestion temperature for the second enzyme cleavage.
• Ensure the volume of the reaction mixture was not reduced due to evaporation during incubation; the increase in salt concentration may reduce enzyme activity. For thermophilic enzymes use a heat block with a hot bonnet, e.g. a PCR cycler.
1.2.2. Improper dilution of conventional enzyme.• Dilute conventional restriction enzymes with Dilution Buffer for Restriction Enzymes (#B19). Restriction
enzymes diluted with this buffer are stable for at least 3-4 weeks at -20°C. • Never dilute enzymes in water or 10X reaction buffer. • Never dilute enzymes in 1X reaction buffer in the absence of DNA.• Use the recommended amount of FastDigest® enzymes, which are experimentally tested on four different
DNA substrates (see Table 1.3 on p.71). 1.2.3. Improper reaction assembly.• The restriction enzyme should always be the last component added to the reaction mixture.• The restriction enzyme may be inactivated if added directly to a 10X reaction buffer. 1.2.4. Excess glycerol in the reaction mixture.• The glycerol concentration in the reaction mixture should not exceed 5%. Thus, the volume of the restriction
enzyme added to the mixture should not exceed 1/10 of the total reaction volume.• Enzymes sensitive to high glycerol concentration include: Alw21I, BpiI, Bsp68I, BspTI, Eco32I, Eco91I, EcoRI,
Hin6I, HinfI, Mph1103I, Mva1269I and NcoI. 1.2.5. Suboptimal DNA concentration. The optimal range of DNA concentration in the reaction mixture is 0.02-0.1 µg/µl.
1.3. Unsuitable DNA template or contaminated DNA solution. If the enzyme is active in the control digest, assay the substrate DNA solution for inhibitory contaminants in
a mixing experiment with control template, e.g. Lambda DNA (dam–, dcm–) (#SD0021). Perform a control digest with two templates, control and sample, in one reaction mixture. Do not exceed the optimal DNA concentration in the reaction mixture (0.02-0.1 µg/µl).
• The sample template is contaminated if neither the control DNA nor sample DNA template is digested (see 1.3.1).• The sample template is not contaminated if the control DNA template is digested but the sample template
is not. Poor digestion of the experimental template is caused by errors in the DNA sequence (see 1.3.2), methylation effects (see 1.3.3) or structure of the DNA substrate (see 1.3.4).
Note Always ensure that the control DNA contains a recognition site for the enzyme present in the reaction. For example, there is no NotI recog-
nition site in lambda DNA.
(continued on next page)
233
1. & CONVENTIONAL RESTRICTION ENZYMES®
1
For patent and license information see p.557 Bulk quantities and custom formulations available upon request
Problem Possible cause and recommended solution
1. Incomplete digestion or no digestion
1.3.1. Contaminants in the DNA solution. • Template DNA may contain residual SDS, EDTA, proteins, salts or nucleases. Repurify the template using a
GeneJET™ PCR Purification Kit (#K0701) or by phenol/chloroform extraction and ethanol precipitation (see p.358). DNA A260/280 ratio should be 1.8-2.0. To remove EDTA and salts, wash the pellet with 70% cold ethanol.
• For reliable and reproducible plasmid miniprep purity, use the GeneJET™ Plasmid Miniprep Kit (#K0502).• For digestion of unpurified PCR products, dilute DNA at least 3-fold in the recommended 1X restriction
enzyme buffer see protocols on p.70 or p.160.• If the template DNA has been purified using silica or resin suspensions, remove all remaining particles by
centrifugation for 10 min at 10,000 rpm and ensure that no resin is carried over while transferring the DNA solution into a new tube.
1.3.2. The substrate DNA does not contain a recognition sequence for the restriction enzyme.• Re-check the DNA sequence and cloning strategy. • Determine if the restriction enzyme selected requires more than one site per target DNA for 100% activity
(see also 1.3.4.3).• Check literature for known site preferences for the restriction enzyme (see also 1.3.4.4).• If the recognition sequence was introduced by PCR primers, verify that the primer sequence contains the
recognition site. 1.3.3. Methylation effects. Restriction enzyme may be inhibited by methylation of the recognition site. • Identify which type of DNA methylation can occur on the recognition site and determine if the methylation
impairs or blocks DNA digestion with the enzyme. See Digestion of Methylated DNA on p.175 and useTables 1.13 and 1.20 on pp.176-181.
If methylation impairs or blocks DNA cleavage:• Propagate your plasmid in an E.coli dam–, dcm– strain (the E.coli GM2163 dam–, dcm– strain; #M0099, is
available upon request with the purchase of any Fermentas product),• Use the REsearch™ engine at www.fermentas.com/research or check the Fermentas catalog for the avail-
ability of a restriction enzyme isoschizomer not sensitive to DNA methylation.• Using of restriction enzyme which requires a methylated recognition sequence (DpnI or SgeI) for digestion
of unmethylated DNA will result in no DNA cleavage. Propagate your plasmid in E.coli dam+ or dcm+ strains (please refer to p.486 for genotype information of some common E.coli strains) to get DNA with methylated DpnI or SgeI recognition sequences. Alternatively, in the case of DpnI, the neoschizomers Bsp143I or MboI can be used to digest non-methylated DpnI recognition sites.
Note When PCR is carried out with standard dNTPs and non-methylated primers the resulting DNA product is not methylated.
1.3.4. Structure of substrate DNA. 1.3.4.1. Supercoiled plasmid DNA. • Use FastDigest® enzymes which are qualified for supercoiled DNA and provided with specific recommenda-
tions for each enzyme (see Table 1.3 on p.71). • For some conventional restriction enzymes, additional units are required to completely digest supercoiled
plasmids (e.g. 5-10 u (1 µl ) of restriction enzyme per 1 µg of DNA). Check notes in the catalog description of the enzyme or refer to the Certificate of Analysis.
1.3.4.2. Proximity of the recognition sequence to the DNA ends. Some restriction enzymes cleave DNA poorly if the recognition site is too close to the end of the DNA molecule. • For FastDigest® enzymes refer to Table 1.3 on p.71 or the product description to determine the effectiveness
of restriction enzyme cleavage at the ends of DNA. • For conventional restriction enzymes refer to Tables 1.10 (p.171) and 1.11 (p.172). • 1.3.4.3. Restriction enzyme requires at least two sites per DNA molecule for optimal activity. Some restriction enzymes such as AarI, BveI, Cfr10I, Cfr42I, Eco57I, EcoRII, LweI, SfiI require at least two
target sites per DNA molecule for efficient cleavage (for more details see Site Preferences by Restriction Enzymes on p.173). If there is only one recognition site per DNA molecule, add a DNA oligonucleotide containing the recognition site.
1.3.4.4. Site Preferences by Restriction Enzymes. The DNA sequence surrounding the recognition site may influence the efficiency of digestion. Some DNA sites
are cleaved slowly or not cleaved at all (for more details see p.173) due to the surrounding sequence. Use ad-ditional units (5-10 u) of conventional restriction enzyme per 1 µg of DNA or determine if an isoschizomer has superior cleavage efficiency (see Table 1.25 on p.207 or REsearch™ engine at www.fermentas.com/research).
Table 1.37. Troubleshooting guide for DNA digestion.
(continued on next page)
234
1. F
astD
iges
t® &
CON
VENT
IONA
L RE
STRI
CTIO
N EN
ZYM
ES
1
www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer
Problem Possible cause and recommended solution
1. Incomplete digestion or no digestion
1.4. Water contains impurities.• Compare your results using commercially available nuclease free, molecular biology grade water, e.g. Water,
nuclease-free (#R0581). Check the quality of the water used in your lab. • Check the pH and conductivity of water. The pH of high quality water should be 5.5-6.0 with a resistance of >18 MΩ.• Centrifuge (10 min, 10,000 rpm) 1 ml of water and check if there is a visible pellet.• Determine if water contains nucleases or bacterial contamination (see 3.2 for control reactions).
2. Unexpected cleavage pattern
2.1. Star activity (relaxed specificity) of restriction enzyme (see p.174 for more details).• Use FastDigest® restriction enzymes. For these enzymes the incubation time without star activity is determined. • Reduce the amount of conventional restriction enzyme (use no more than 10 u).• Use the recommended reaction buffer.• Ensure that the glycerol concentration in the reaction mixture does not exceed 5%. • Reduce the incubation time. For FastDigest® enzymes – refer to Table 1.3 on p.71 for maximum incubation times.• Ensure the volume of the reaction mixture was not reduced due to evaporation during incubation. The
resulting increase in glycerol concentration may cause star activity. 2.2. Contamination with another restriction enzyme. The restriction enzyme or buffer may be contaminated with another restriction enzyme due to improper
handling. Use a new tube of enzyme and/or buffer.2.3. Contamination with another substrate DNA. The sample DNA may contain a mixture of two or more different DNAs. Prepare new sample of DNA. • For plasmid DNA preparation pick one isolated colony of recombinant E.coli grow cells and purify plasmid
with GeneJET™ Plasmid Miniprep Kit (#K0503).• For PCR products: check the product purity on an agarose gel. If necessary, purify the PCR product prior to
digestion with GeneJET™ Gel Extraction Kit (#K0692) or Silica Bead DNA Gel Extraction Kit (#K0513).2.4. Incomplete DNA digestion (see 1). 2.5. Gel shift (see 3.1).2.6. Unexpected recognition sites in template DNA. Newly generated target sites in constructed DNA may be overlooked. Re-check your DNA sequence and
cloning strategy. Refer to Tables 1.21, 1.22, 1.23 or 1.24 for Newly Generated Recognition Sequences (pp.182-206) to identify all the cleavage sites present in the substrate DNA.
3. Diffused DNA bands
3.1. Gel shift. Enzyme that remains bound to the substrate DNA will affect the electrophoretic mobility of the digestion products.
FastDigest® Restriction Enzymes BspMI (BveI), HgaI (CseI), AcuI (Eco57I), FokI, MboII, TauI, TspRI (TscAI) and con-ventional restriction enzymes AarI, AloI, BdaI, BseXI, BveI, CseI, Eco57I, Eco57MI, EcoRII, FaqI, GsuI, Lsp1109I, LweI, MboII, MnlI, SchI, TauI, TscAI, TsoI and TstI are particularly prone to remaining bound to the substrate DNA. This will result in a band or smear above the expected band (see picture below). Heat the digested DNA for 10 min. at 65°C in the presence of 6X DNA Loading Dye & SDS Solution (#R1151) or 0.2% SDS prior to electrophoresis.
M – GeneRuler™ DNA Ladder Mix (#SM0331).1 – 0.5 µg λ DNA prepared for loading with 6X DNA Loading Dye (#R0611).2 – 0.5 µg λ DNA prepared for loading with 6X DNA Loading Dye & SDS Solution (#R1151).3 – 0.5 µg λ DNA digested with TsoI (#ER1991), probe prepared for loading with 6X DNA Loading Dye (#R0611).4 – 0.5 µg λ DNA digested with TsoI, probe prepared for loading with 6X DNA Loading Dye & SDS Solution (#R1151).
M 1 2 3 4
3.2. Contaminated reagents. Any restriction digestion reaction components may become contaminated with nucleases due to improper handling
or storage. Nuclease contamination causes DNA degradation, which appears as diffused DNA bands on a gel. Perform four control reactions in order to check for the nuclease contamination: I – without restriction enzyme, II – with a new vial of buffer, III – without restriction enzyme, with a new vial
of buffer, IV – with commercially available water e.g. Water, nuclease-free (#R0581).• Contaminated sample DNA (diffused bands in all controls). Prepare new DNA sample (re-purify DNA).• Contaminated enzyme (diffused bands in controls 2 and 4). The enzyme may become contaminated due to
improper handling. Use a new vial of enzyme.• Contaminated buffer (diffused bands in controls 1 and 4). Bacterial contamination of the reaction buffer will
cause DNA degradation. Use a new vial of buffer. Store all buffers at -20°C.• Contamination of both enzyme & buffer (diffused bands in controls 1, 2 and 4). Follow the recommendations given above.• Contaminated water (diffused bands in controls 1, 2 and 3). Bacterial or DNase contamination in improperly handled water
will cause DNA degradation. Use commercially available nuclease-free molecular biology grade water (e.g. #R0581).
Table 1.37. Troubleshooting guide for DNA digestion.