PCR problems

Jose de las Heras jose.delasheras at virgin.net
Thu Aug 5 14:50:45 EST 2004


"Kyle Legate" <legatek at hotmail.com> wrote in message
news:2nck1dFtvkpfU1 at uni-berlin.de...
> rahul.ranjan at biocon.com wrote:
> > At higher PCR cycles we observe degradation in all the samples
> > (including the blank) in the form of smear (streaks). Although at
> > lower cycles we observe a sharp band. Higher cycles are need to
> > obtain an amplification of 1pg DNA (genomic DNA).
> >
> What could possibly be degrading in your blank. I think this is a larger
> problem.

Yep, it looks like you're getting some contaminant which has multiple
homologies (or close enough, at the annealing temperature that you use) to
your primers... with lower cycles there may not be enough non-specific
amplification to be visible on the gel... but increase the cycles and it
becomes apparent.

Non-specific amplification with high cycles is not uncommon in genomic PCRs,
by the way. Aim for the highest annealing temp you can use, and don't overdo
the number of cycles justto obtain more product. It's best to run more
reactions.

Jose





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