PCR of GC-rich sequence

[Martin Hoagland]

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
<hoa...@pop.uky.edu>