Restriction enzymes
- Created by: kpritchard16
- Created on: 19-12-18 17:54
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- Restriction enzymes
- They were orignally identified to protect bacteria from phage infection whilst cellular DNA protected by methylases
- EcoR1 from E.coli
- BamH1 from B. amyloliqueraciens
- Pvu1 and Pvu11 are different enzymes from same strain
- They are able to cut DNA molecule at specific sequence, same sequence in any DNA
- Properties
- DNA recognition sequence is usually 4-8bps
- Recognition sight may be ambiguous
- PuGCGCPy or CCTNAGG
- Cuts may be blunt or sticky ends
- Average distance between cutting sites is determined by how long the recognition sequence is and probability of finding each nucleotide
- Different classes
- Blunt ends
- Sticky ends
- 5' or 3' overhands
- Need combatible ends with same overhand except for blunts, can ligate any two blunts together
- Harder with blunt ends as no overlap for base pairing
- Optimal conditions
- pH = 7,5 8.0, 8,5
- Reducing reagent = DTT
- Carrier protein = BSA
- Divalent cations = Mg2+
- Temperature = 37, 25, 60
- Salt conc = 20mM, -150mM
- They were orignally identified to protect bacteria from phage infection whilst cellular DNA protected by methylases
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