High G-C PCR...help.

[Eric C. Anderson]

> 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
> language...nothing.
> It has a very high (about 75%) G-C content...is this a problem?
> Anybody else had this experience?

steve,

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.

good luck,

eric