Error-prone PCR in directed evolution experiments

Trond Erik Vee Aune teva at online.no
Wed Jun 6 10:42:02 EST 2001


Hi

I am about to start directed evolution on a procaryot transcription
factor, and I was planning to use error-prone PCR as the method to
generate mutants of the gene. I have heard that stratagene has released
a kit for this procedure, does anyone here have any experience with this
kit?

The problem, as I see it, is determining how many point mutations I
want, setting up the reaction, and afterwards, finding out how many
point mutations I actually got. This obviously calls for a good
screening procedure. My primary objective is increased expression from
the promotor, and the secondary is to redesign the trancription factor
to accept different inducers. We have cloned the amp gene downstream of
the promotor, so screening should be faily easy.

Does anyone have any ideas, suggestions, help and hints? Especially the
optimization of the PCR reaction seems time-consuming and tricky. 

Regards,
Trond Erik Vee Aune




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