Team:Warsaw/Calendar-Main/13 October 2009
From 2009.igem.org
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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