svetlov at oncology.wisc.edu
Wed Mar 18 10:26:21 EST 1998
In article <bbeitzel-1603980809000001 at idl04.salk.edu>,
bbeitzel at biomail.ucsd.edu wrote:
> Just wondering if anyone had any suggestions about mutagenesis. I have a
> cDNA that I would like to mutagenize to give me a pool of different
> mutants (point mutants and small deletions.) Any recommendations as to
> the best way to go about doing this?
No random mutagenesis protocol will reliably generate small deletions,
point mutations can be easily generated by Taq-driven PCR in presence of
Mn++ and/or high dNTP concentrations. Use a lot of template and a few
cycles (20) to avoid propagation of a few mutations "early" in
amplification throughout the population of PCR products. More laborious
techniques include addition of an excess of one of the dNTPs or dITP, but
these also have some bias in the kind of mutations you are getting.
McArdle Lab for Cancer Research
1400 University Ave.
Madison, WI 53706
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