Smear PCR product from high GC template

Liz Parks liz_parks at ncsu.edu
Tue Feb 9 10:14:25 EST 1999


I'm not sure if all that is true in this case.  With such a high GC
content, the problem may be that your template is not completely
denatured in your denaturation step.  I have found that Pfu polymerase,
used instead of Taq can help.  Pfu will allow you to denature at a
higher temperature (97C) than you can with Taq.  I'd also try adding an
initial 5 minute denaturation before cycling starts (you don't need to
wait to add Pfu as in hot start).    Formamide, as suggested, may help
with denaturation.  Touchdown PCR would not necessarily help.  I would
not use a conc.of Mg above 1.5mM, as this will increase smearing.  Good
luck!

Liz

--
Liz Parks
North Carolina State University
Plant Pathology
Raleigh, NC  27695





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