help with PCR trouble shooting

[Yu Wai Chen]

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.

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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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