Mutagenesis:Advice Needed

Rick Davis richard..davis at worldnet.att.net
Tue Oct 28 15:39:24 EST 1997


Our lab is trying to replace a 66 bp region of the T.gondii HGPRT gene
with the corresponding homologous region of human HGPRT sequence.
I've utilized both the Stratagene QuikChange kit and the megaprimer
technique to accomplish this without success. The primers we designed
have 15 bp of sequence 100% homologous to T.gondii HGPRT at either
end and the 66 bp of human sequence in the middle. Is 15 bp of
homologous sequence at either end of the primer sufficient for
annealing of the primers to target, given the resulting large 66 bp
loop out? Can anyone suggest guidelines for primer design and PCR
annealing conditions when attempting loop out constructions like this?
Also, do any of the commercially available mutagenesis kits perform
better with these kinds of constructions? TIA



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