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.

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