Strange PCR buffer problem

Mikael Niku mikael.niku at nospam.vetmed.fi
Wed Jan 30 06:15:51 EST 2002


Hello!

I have a strange problem with my PCR buffer (or perhaps
combination of this and my gel loading buffer or
electrophoresis buffer).

I first thought there was something wrong with the
PCR reaction. But now I found out that if I load just
1x PCR buffer (with 1x loading buffer) on the gel, I get a
pretty strong band traveling at about 100 bp and leaving a
smear reaching to the wells, fainting towards the wells.

I don't get this with the whole PCR reaction mix or with
any of the other components. Also the loading buffer doesn't
interfere with the DNA size standard if added to it.

The PCR doesn't work or works only poorly (I can't
be absolutely sure if it's really connected with the buffer
problem, but occurred in the same time).

The reagents I'm using:

* PCR buffer - a standard commercial buffer, containing
  Tris-HCl, MgCl2, KCl, Triton

* agarose gel electrophoresis buffer: standard 1X TBE

* loading buffer: home-made buffer containing Ficoll,
  EDTA, SDS and bromphenol blue; stored at RT

All the reagents have been working OK before in identical
conditions. The only change is (unavoidably) in their age.
Using a fresh unopened 10X PCR buffer tube doesn't help.
I haven't tried preparing fresh loading buffer or
fresh TBE yet but they don't look in any way strange.
And I repeat, the strange thing occurs ONLY if I have
the PCR buffer in the gel.

I can't really understand this. It happens like if there
was some kind of reaction between PCR buffer and
electrophoresis stuff... but why? Any ideas?

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   Mikael Niku             URL: www.helsinki.fi/~mniku/
   University of Helsinki  Dept. Basic Veterinary Sciences
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                                              - Gandhi
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