>High GC PCR"

Dr. Peter Gegenheimer PGegen at UKans.nolospamare.edu
Tue Nov 9 17:13:25 EST 1999


On Tue, 9 Nov 1999 00:19:33, "Don Walthers" <gene_jockey26 at yahoo.com> wrote:

ð Lowering the annealing temp and increasing the annealing time plus the
ð addition of 5% DMSO helps my GC-rich PCR (Myxococcus). My primers usually
ð have Tm's of about 85-90 degrees. I usually use 95 1', 50-55 2' and 72 @1'/kb 
ð expected product.

Betaine (0.5 - 1.0M) is also said to help. We've used it successfully, but I 
don't recall that it was absolutley necessary. 

Also, if the Tm of your primers is 85-90C, then a normal annealing temperature
would be about 75C or so. You might try a 2-step PCR (omit the annealing step;
run the elongation at 72 to 75C). You may well be losing specificity by 
annealing at such a low temperature!

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