PCR SMEARING

tfitzwater at gilead.com tfitzwater at gilead.com
Mon Nov 13 12:27:53 EST 2000


>Philip O'Brien (obrien at central.murdoch.edu.au)
>Thu 09 Nov 2000 - 06:55:26 GMT


>We have been getting a lot of smearing in our PCR reactions. Sometimes we
>get a discreet band, but most of the time its a smear. We get this with
>different primer pairs so it dosent't appear to be confined to one pair.
>The reactions are all set up on ice to minimize aberrant priming before
>thermalcycling. I would be grateful if anyone could give me information on

>how to eliminate the problem.


>Philip O'Brien
>Division of Science
>School of Biological Sciences & Biotechnology ,
>Murdoch University,
>Murdoch WA 6150,
>AUSTRALIA


If the smear occurs on native gels but resolves on denaturing gels, then
the problem is due to too much template or too many cycles for the amount
of template.  As the molecules of product in the reaction exceeds the
number of molecules of enzyme, the strands can form quadruplexes (and n+2
in decreasing frequency) that travel as smears on native gels.  Completely
denatured material will travel as a single band on a denaturing gel, while
incomplete denaturation will result in 2-5 very sharp bands.


Tim Fitzwater


Principal Research Associate


Gilead Sciences





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