High G-C PCR...help.
Eric C. Anderson
e-anderson at ski.mskcc.org
Thu Apr 18 12:35:19 EST 1996
> I need advice on PCR...I've tried many times to amplify
> a 1.2 Kb insert with multiple primers, different enzymes,
> different templates, different TM's, and lots of bad
> It has a very high (about 75%) G-C content...is this a problem?
> Anybody else had this experience?
try either or both of the following:
1. increase the amount of dGTP and dCTP in your reaction mix by about 50%.
2. use a 2-step cycling program that has only a denaturing step and an
extension step. when i need to do this kind of PCR i usually use 94oC for
30s --> 70oC for 1-2min.
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