help, gc rich pcr.

Steven Goldberg goldberg at
Tue Sep 5 07:07:45 EST 1995

I have worked with Actinomycetes DNA (ca. 72% GC) and found the 
following conditions may help when PCRing:

1.  Include 5-10% DMSO to relax the DNA (double the amount of enzyme 
2.  Try "hot start" to increase specificity.
3.  Test differing Mg++ concentrations from 1-5 mM.
4.  Important control:  Test each primer in a separate reaction.  You 
get a lot of false positives with a single primer due to non-specific 
homology to GC-rich DNA.

I'm sure there are other modifications, but these have been the most 
important for our work.  Unfortunately, there are times it seems to be 
impossible to get a single PCR band when using high GC DNA, so it may be 
necessary to sort through the two or three best candidates.

Hope these suggestions help.


More information about the Methods mailing list