From owner-biotechniques@net.bio.net Thu Nov 05 22:00:00 1998 Path: biosci!biosci!not-for-mail From: Martin Hoagland Newsgroups: bionet.journals.letters.biotechniques Subject: PCR of GC-rich sequence Date: 6 Nov 1998 06:13:23 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 24 Sender: daemon@net.bio.net Approved: mmccarthy@BioTechniques.com Distribution: world Message-ID: <71v063$dmc@net.bio.net> NNTP-Posting-Host: net.bio.net Dear Biotechniques, I am currently working on constructing a clone but have ran into problems as most graduate students do. The region I am seeking to amplify is GC rich and the plasmid DNA could be forming tertiary complexes preventing PCR amplification. I have consulted the texts and tried increasing MgCl2 concentration as well as DMSO and glycerol. These modifications have not yielded my expected product. I have used a five minute denaturation step in my PCR at 95 degrees Celsius. I was wondering if I might denature it with NaOH. Will this interfere with taq DNA polymerase activity at an incresed pH and salt concentration? Sincerely, M. Hoagland .