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Digesting DNA with restriction endonuclease Mix the DNA solution and reaction buffer (Total volume: Y ml) DNA solution X m l( 5 m l) 10x reaction buffer 1/Y m l( 1 m l) Restriction Enzyme 1 m l( 1 m l) Add ddH 2 O to Y m l( 3 m l) - PowerPoint PPT Presentation
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Digesting DNA with restriction endonuclease
1. Mix the DNA solution and reaction buffer (Total volume: Y ml)
DNA solution X l ( 5 l) 10x reaction buffer 1/Y l ( 1 l) Restriction Enzyme 1 l ( 1 l) Add ddH2O to Y l ( 3 l)
2. Incubate at 37 for 1 hour (depend on restriction en℃zymes)
3. Argarose gel electrophoresis
Recombinant DNA Technology
Gene cloning or Molecular cloning
1. The recombinant DNA construction
2. Transformation
3. Identification
The recombinantDNA construction
Transformation
Identification
Restriction Endonucleases
Restriction Endonucleases
Restriction Endonucleases
Restriction Endonucleases
Restriction Endonucleases Nomenclature
1. Eco RI Escherichia coli R2. Hpa I Haemopophilus parainfluenzae
Palindromic sequence Types of cut ends
1. 5’ phosphate extension2. 3’ hydroxyl extension3. Blunt end
Fig 4.2 Palindromic sequence
Fig 4.3
Table 4.1
Isochizomer
Different restriction enzymes recognize different sequences
Cleave within the same target sequences
Sau 3A, Mbo I & Bam HI (5’…GATC…3’)
SalI & XhoI (5’-GTCGAC- & 5’-CTCGAG-)
Fig 4.4
Fig 4.5
Fig 4.6
Ligases E coli T4
Fig 4.7
Table 4.3
Klenow fragment
3’ to pyrimidine
3’ to guanine
XceI RCATG/Y NspI RCATG/Y BstNSI, NspI
XcmICCANNNNN/
NNNNTGG XcmI CCANNNNN/NNNNTGG
XhoI C/TCGAG PaeR7I C/TCGAGPaeR7I, Sfr274I, SlaI, S
trI, TliI
XhoII R/GATCY BstYI R/GATCYBstX2I, BstYI, MflI, Ps
uI
XmaI C/CCGGG SmaI^ CCC/GGGCfr9I, SmaI^, TspMI, X
maCI
Enzyme Sequence Enzyme Sequence Other Isoschizomers
AanI TTA/TAA PsiI TTA/TAA PsiI
AarI CACCTGC (4/8)
AasIGACNNNN/
NNGTC DrdI GACNNNN/NNGTC DrdI, DseDI
AatI AGG/CCT StuI AGG/CCTEco147I, PceI, SseBI, S
tuI