help, gc rich pcr.
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
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.
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