Team:Warsaw/Calendar-Main/13 October 2009

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Mutagenesis of cI

Kuba

Task: Obtain a functional, termosensitive version of cI

Method used:

  • inverse PCR
  • gel purification
  • ligation
  • DpnI digest
  • transformation into E. coli (top 10)

The mutagenic PCR was preformed on both - the bistable switch and cI on a pSB vector

Day1:

  • PCR
- primers used cIR(mut) and cIL(mut)
- initial denaturation: 95 oC, 5 min
- denaturation: 95 oC, 30 sec
- annealing: 46 oC, 45 sec
- elongation 72 oC, 9 min
- 35 cycles
  • gel purification
  • PNK treatment of the purified product (over-night)



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