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PCR amplification from yeast genomic DNA

Hoffmacs Hoffmacs at HERMES.BC.EDU
Sun Jul 31 02:57:15 EST 1994


Janet,
Many labs now just add a small portion of a freshly grown colony (50,000 cells)
directly to the reaction.  How do you know this problem is specific to the
genomic DNA?  Does it work for plasmid?  Do you see anything for the genomic
amplification?  There are several possibilities as to what went wrong.  These
are not necessarily associated with your DNA prep.  You may also want to try
other enzzymes such as VENT from New England Biolabs or Pfu from Stratagene (we
use VENT).  Good luck.
Charlie Hoffman
"I'd rather be fission."
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To: yeast at net.bio.net
From: Janet Shaw on Fri, Jul 29, 1994 6:32 PM
Subject: PCR amplification from yeast genomic DNA
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To: yeast at net.bio.net
From: shaw at bioscience.biology.utah.edu (Janet Shaw)
Subject: PCR amplification from yeast genomic DNA
Followup-To: bionet.molbio.yeast
Date: 29 Jul 1994 22:55:31 GMT
Message-ID: <shaw-290794155200 at ls-37.biology.utah.edu>
NNTP-Posting-Host: ls-37.biology.utah.edu

We've been PCR amplifying fragments of the yeast genome for various cloning
purposes.  Recently, we've run across several primer sets that don't work
well, work sporatically or don't work at all on yeast genomic DNA as
template.  Does anyone have suggestions or a reliable protocol for
amplification of fragments from yeast genomic DNA?  (We are currently using
an air cycler in 10ul reaction tubes but have access to Perkin-Elmer type
thermocyclers as well).
Thanks,
Janet Shaw
University of Utah
shaw at bioscience.utah.edu  





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