checking site-directed mutagenesis

orsolya.benedek at orsolya.benedek at
Wed Jun 26 10:03:19 EST 2002

 I tried to do site-directed mutagenesis with the PCR-based megaprimer method using two consecutive PCR reaction.  My template to mutate was a Y.pestis plasminogen activator gene cloned into pUC 19.The proposed mutation did not introduce or remove any new restriction sites so I did digestions with enzymes producing two fragments: the vector pUC 19 and the insert.  Mutated clones gave exactly the same fragments as the original wild-type template but were  functionally inactive and they did not seem to express the protein. Why could this happen? I'm still waiting for sequencing which goes awfully slowly at our university. I'm just wondering how much is worth waiting for sequencing or restart everything again in a different way.

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