Team:Warsaw/Calendar-Main/13 October 2009

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: - 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
 * PCR


 * gel purification
 * PNK treatment of the purified product (over-night)