PCR from genomic dna

biogeekgrrl biogeekgrrl at my-deja.com
Thu Jun 8 15:54:08 EST 2000


In article <392EE3F3.9C4C5096 at tribe.ulm.edu>,
  vaidyaks at tribe.ulm.edu (Kedar Vaidya) wrote:
> I have been trying to get a specific part of the dna amplifies by
> PCR..the primers seem pretty ok. they work when i pcr from plasmids but
> i get no product from the genomic dna...any suggestions?

In my experience, failure to amplify genomic DNA is most often due to
insufficient denaturation.  I always use 5% DMSO in genomic PCR, and perform
a 3 minute hot start.

Second most common culprit is contamination in the DNA.  Try a dilution
series-- ie 25 ng, 50ng, 100ng and so on.  As long as the template isn't too
dirty, one of those conditions should work.  I routinely perform 50 ul
reactions using ~100 ng tail DNA prepped by good ol' phenol chloroform
extraction, and have very few problems.  Just take care in the preparation of
your samples, and you should be okay.

Best of luck. -- "Like most scientists, Gabe was oblivious to the fact that
no one gave a rat's ass about research unless it could be expressed in terms
of dollars." -Christopher Moore


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