Smear PCR product from high GC template

Burkhard Hassel b.hassel at dkfz-heidelberg.de
Tue Feb 9 08:46:09 EST 1999


Hi Andrew!
too many cycles?
too much polymerase?
annealing temp too low (I wouldn't use touchdown) ?
tried formamide (Sarkar et al. NAR 18(24) p. 7465)?
tried alternative polymerases (e.g. 1/15 mixture of Pfu pol and Taq pol)?

smear generally comes from too unspecific + too much (template or product)
yours, burki

Andrew Leung wrote:

> Hi all,
>
>     I am attempting to amplify a template which has 70%GC content. The
> problem is that I got smear products in gel analysis. I have tried to
> varying PCR condition including Mg++(1 to 2.5mM), annealing temp(touchdown),
> and addition of DMSO (0-10%). But the problem has not be solved.
>
>     Is there any possibility to account for the smear
>
> Andrew






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