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help with PCR trouble shooting

Michael Cooley szcooley at chip.ucdavis.edu
Fri Oct 18 16:56:14 EST 1996

Yu Wai Chen (wai at dta.med.harvard.edu) wrote:
: Dear all,
: 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?
: Thank you.
: ===================================================================
: 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
: ===================================================================

How much DNA did you put into the reaction. From the sound of it I would
say quite a lot. Also did you do anything to remove the old primers from
the first reaction? If you had you could not have amplified the 1500 bp
fragment again. Additionally you may want to try touchdown PCR. Later,
Mike Cooley

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