Team:Warsaw/Calendar-Stage1/14 September 2010

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Mutagenesis of MinC

Kuba

Directed mutagenesis was preformed on pSB-MinC in order to remove an additional PstI site. Two primers were used: MinCmutF (TAATTGCAGTCGCGCCGC) and MinCmutR (GTCGAAAACGCTTTGACCG). PCR was run with Yellow Pfu Polimerase (Eurex) in a standard reaction mix.

PCR conditions were as follows:

• initial denaturation 95 ^oC; 3'
• denaturation 95 ^oC; 30 s
• annealing 51 ^oC; 30 s
• elongation 72 ^oC<; 7'
• x 30 cycles
• 72 ^oC; 10'

PCR product was digested with DpnI amd purified by agarose electrophoresis.