help, gc rich pcr.

Steven Goldberg goldberg at bms.com
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 
used). 
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.

Steve





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