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The Gateway® Cloning SystemHow to generate an entry clone
Contents
• Options for entering the Gateway® system
• Defining the BP Clonase™ reaction
• Defining the LR Clonase™ reaction
• Description of Ultimate™ ORF collection
• Description of Vector NTI Advance™ software for in silico cloning
Invitrogen Proprietary & Confidential2
The Gateway® Reactions
LR clonase
++BP clonase
attL1 attL2
EntryClone
KanR
ExpressionClone
attB1 attB2
AmpR
attR1 attR2
ccdB
DestinationVector
AmpR
attP1 attP2
ccdB
DonorVector
KanR
Invitrogen Proprietary & Confidential3
Different ways to generate the entry clone
2. TOPO® Cloning
TOPO®BP Clonase™
1. BP Cloning
PCR Product
+TOPO-ActivatedEntry Vector
L1 L2
Gene+attB PCR Product
B2GeneB1Donor Vector
P2ccdBP1
Entry Clone
L2GeneL1
+digested DNA Fragment
GeneB1digested Entry Vector
L2L1
4. Pre-made entry clone5. Custom-made entry clone
Ligase
3. Restriction/Ligase Cloning
ORF Collection
L2ORFL1
Invitrogen Proprietary & Confidential4
1. BP Cloning – The Reaction
90-99% correct cloneson Kan plates
geneattB1 attB2
+attP1 attP2
ccdB
D o norVector
K anR
+
gene
attL1 attL2
EntryC lone
K anRccdBattR1 attR2
BP Clonase™
Invitrogen Proprietary & Confidential5
1. BP Cloning - Primer Design for PCR
• GGGG and the attB1 sequence must be added to the 5’-primer (sense)
• GGGG and the attB2 sequence must be added to the 3’-primer (antisense)
attB1
5’ – GGGGACAAGTTTGTACAAAAAAGCAGGCTNNN…
attB2
5’ – GGGGACCACTTTGTACAAGAAAGCTGGGTNNN…
Gene SpecificPrimer Sequence
Invitrogen Proprietary & Confidential
*After overnight incubation
1. BP Cloning – Some Examples
Size (kb) PCR DNA(fmol)
PCR DNA(ng)
Colonies/lTransformation
CorrectClones/Total
Clones Examined
0.261538
37.5
12232815
10/10
1.01538
1025
5071447
49/50
1.41538
1435
271683
48/50
3.41538
3485
478976
9/10
4.61538
46115
190195
10/10
6.91538
69173
30 (235)*54 (463)*
47/50
10.17.5
37.550.5252.5
16 (112)*42 (201)*
15/16
TyrosineKinase
Transferrin ReceptorTarget Template: EIF4e b-Adaptin MAP4
attB-containing primers: + + - - + + - - + + - - + + - - + + - -
Platinum® Pfx DNA Polymerase
Platinum® Taq DNA Polymerase
High Fidelity
Platinum® Taq DNA
Polymerase
Total RNA isolated from HeLa cells, first strand cDNA synthesized using THERMOSCRIPT™ RT.
1. BP Cloning – RT-PCR Using attB-Containing Primers
Invitrogen Proprietary & Confidential8
2. TOPO® Cloning -TOPO®TA
Invitrogen Proprietary & Confidential9
2. TOPO® Cloning – Directional TOPO®
Invitrogen Proprietary & Confidential10
3. Restriction/Ligase cloning
• Use when there are convenient sites to cut insert out of another plasmid
• Must cut out ccdB gene by using one of four RE sites flanking the ccdB
• Reading frame of insert must be considered, as well as downstream expression elements
• Various reading frames of pENTR vectors are available
pENTR™ 1A Vector in reading frame 0
pENTR™ 2B Vector in reading frame +1
pENTR™ 3C Vector in reading frame +2
pENTR™ 4 Vector in reading frame 0; modified polylinker from 1A
pENTR™ 11 Contains both an E. coli (SD) and eukoryotic (Kozak) ribosome binding site
Invitrogen Proprietary & Confidential11
4. Pre-existing ORF collection
16,272 human ORFs (Oct 2006
release)
Amber stop codons
Sequence verified
Ready to use in LR reactions
http://orf.invitrogen.com/cgi-bin/ORF_Browser
Invitrogen’s Ultimate™ ORF collection
Invitrogen Proprietary & Confidential12
5. Custom Gene Synthesis
• Quick and cost-effective• No PCR amplification necessary• 100% accuracy (sequence verified)• Optional codon optimization for
expression
Invitrogen Proprietary & Confidential13
In silico cloning using Vector NTI AdvanceTM 10.3
Primers for PCR reaction
Cloning Strategy
DNA of interest
Invitrogen Proprietary & Confidential14
Gateway® Summary
Gene
Gene
Entry C lone
O RFcollection
PC R Library
Genesynthesis
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