help with PCR trouble shooting
Yu Wai Chen
wai at dta.med.harvard.edu
Fri Oct 18 11:22:11 EST 1996
I am new to PCR. I am using PCR to do "overlap extension" as an intermediate
step in site-directed mutagenesis. Basically, I have 2 fragments of dsDNA which
has an overlapping complementary sequence of 25 bp, both containing the mutation.
I need to use the heteroduplex with 3' overhangs as templates and generate
full-length dsDNA as for a subsequent amplication.
My two fragments are 300bp and 1500bp respectively. I did
PCR for 30 cycles (92C, 1 min; 50C, 1 min; 72C, 1.5min) using vent polymerase.
What I see, after the reactions is no expected 1800 bp band. The 1500 bp
starting material band is still there. That means the overlap extension was not
working. Surprisingly, the 300bp band has gone. Instead, I saw a band of very
high molecular weight. It stays right at the edge of the gel well. It looks
like the 300bp startng material has aggragated itself. Can somebody tell me
what's going on and perhaps suggest any tricks to get around the problem?
Yu Wai CHEN, Ph.D. Department of Microbiology
................................. & Molecular Genetics
tel: (617) 432-1931 Harvard Medical School
fax: (617) 432-0115 200 Longwood Avenue
email: wai at dta.med.harvard.edu Boston MA 02115
www: http://dta.med.harvard.edu/wai.html USA
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