random gene mutagenesis

[Linda Foit]

Hi!

Does anybody know a good protocol for megaprimer-extension-PCR?

I am trying to mutate a gene on a expressionplasmid by generating megaprimers (about 1000 bp) with the Genemorph II mutagenesis Kit. I gel-purify those megaprimers and use them as primers for amplification of the whole plasmid in a second PCR round. 
Unfortunately, I get only a very small amount of PCR-product in the second PCR-round (nearly no colonies on selective plates after transformation). 

Does anybody have suggestions for improving the PCR-conditions for the mega-primer-extension PCR? 


Thank you very much!





Linda Foit
PhD student

Dept of Molecular Cellular and Developmental Biology
University of Michigan