Site-directed PCR mutagenesis

[Hans Kiefer]

Dear experts,

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.

Cheers,

Hans