Site-directed PCR mutagenesis
hans at biokemi.su.se
Thu Mar 28 05:51:09 EST 1996
I would like to know what your favourite method for PCR-based
site-directed mutagenesis is, and why. I started recently using an
asymmetric PCR with a single mutant primer (NAR 18, 7443, 1990), and
it works in most cases, but not in all. Sometimes there is no PCR
product in the second step. I switched from Taq to Pfu polymerase,
because I wanted to get rid of all the extra A´s added by Taq (which
was successful). But Pfu seems to be more sensitive towards the
optimal Mg concentration. I also tried glycerol and DMSO as
cosolvents, but it didn´t seem to improve anything.
If this topic has been discussed here earlier, maybe there is a
summary which you could e-mail to me. Otherwise, it would be
interesting to hear your opinions, and I promise to post a summary.
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