Difficult PCR

Marek Treiman Marek.Treiman at med.kuleuven.ac.be
Thu Jun 22 05:02:37 EST 2000


Thanks everyone for the input!

Marek

Marek Treiman wrote:

>     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






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