Restriction enzymes

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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

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