site directed mutagenesis
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
J. E. Graham
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