Difficult PCR

[Marek Treiman]

    Hi everyone,

I have been trying for almost 2 months now to PCR a promoter region
from  human DNA, approx. 3.4 kb. About 25% of it on the 3' side is very
GC-rich. The reverse primers I have been using had all a HindIII
overhang attached. I have used several primer pairs, some of the oligos
as long as 35 bases, trying to go to as high annealing temperatures a
possible. Tried Boehringer Expand Long Template system and Clontechs
Advantage GC-PCR kit. Both step-down and "flat" temperature approaches
failed. Always lots of non-specific priming (until I pushed the
Mg/temperature so much that no priming occurred at all). At this point,
I guess I have 2 questions to PCR experts: 1. is there a method, a
"trick", a condition that I definitely should try and haven't? 2. Does
it happen that in some situations, PCR just simply fails and one has to
live with it?

Advice, comments, informed remarks will be very wellcome!

Cheers (muted),
Marek