High G-C PCR...help.

Paul N Hengen pnh at cockleberry.ncifcrf.gov
Fri Apr 19 11:29:46 EST 1996


Eric C. Anderson (e-anderson at ski.mskcc.org) wrote:

> 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?

: 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.

Also have a look at this article:

@article{Hengen1996Jantibs,
author = "P. N. Hengen",
title = "Methods and reagents - Cycle sequencing through
{GC}--rich regions",
journal = "Trends in Biochemical Sciences",
volume = "21",
number = "1",
pages = "33-34",
month = "jan",
year = "1996"}

Good Luck!

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