site directed mutagenesis

the End jgraham at bronze.ucs.indiana.edu
Fri Sep 2 10:37:57 EST 1994


Don't waste you time with any kit, delve into the literature as that's 
where you will be headed as soon as you complete the steps on the 
"card" and don't find your desired mutants.

After trying many of the basic approaches, I find that you can almost 
always obtain your mutant in a few days by incorporating a mismatch 
primer by PCR. Limit your cycle number to less than 15 and increase the 
template plasmid level to 200 ng / 100 ul rxn. You will not create 
additional unwanted changes.

Should your site lie within a region devoid of flanking restriction sites,
you can either add sites by further mismatch in the primers if you are 
altering a protein target, or otherwise try the two PCR "megaprimer"
method where the entire PCR product is used as a "primer" in a second
PCR with a third primer which encompasses a restriciton site. Keep the 
first target as small as possible (<~500 bp) for good results with this 
approach.

Good luck,

Jim
J. E. Graham 
Institute for Unrestricted Information Exchange
Ameriscam Inc. Liberal, KS 



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