PCR Problems

Rod Pennington rodpenn at brcsun0.tamu.edu
Fri Jan 31 14:41:04 EST 1997

Brian Foley wrote:

> > We are using 21-mer primers (that have been re-made 3 times, by two
> > different oligo suppliers) to amplify a 290 kb sequence of the target DNA.
> > These primer sequences were carefully designed and have worked fine in
> > the past.  The problem is that recently, when running the products out on a
> > 1% agarose gel, we get a smear that is generally larger (3 - 10 kb) than the
> > anticipated product.....

> > *  Richard K. Meister                  Email:  Meister.1 at osu.edu  *

> ... Perhaps your PCR works great, but their is
> something wrong with running your gels.  I suspect that
> you would have noticed that problem though, because your
> molecular weight markers would smear as well as the
> PCR product. 
> |Brian T. Foley                btf at t10.lanl.gov                      |

Brian may well be correct.  I doubt very seriously that such a problem is
the result of amplification of a contaminant DNA.  For one thing, if you
(RKM) are intending to amplify a short (290 bp, I assume you meant 
bp instead of kb) fragment, you are probably using an elongation step
of 1 minute or less.  This isn't enough time to amplify a 3-10 kb target
sequence.  If I recall correctly, Taq's "maximum speed" is about 1 kb/min.

Could there be some junk in your mineral oil overlay that is mixing with
your samples and screwing up the gels?

Rod Pennington
Temple, TX

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