PCR rxns give smears. Any advice?

Brain Foley brianf at dna.uvm.edu
Tue Dec 1 17:32:58 EST 1992


In my experience a smear of amplified DNA usually indicates too much
TAQ, too much template, or too many cycles.  Have you switched to a
new lot of TAQ that may have more units of activity per microliter?
Try removing a 10 microliter aliquot after 15 and 20 cycles and running
that on a gel.  I have seen reactions that looked great at 15 cycles
end up with just a big smear after 25 cycles.  Also try calibrating
the amount of TAQ.  Try 0.5, 1.0 and 2 units per 100 microliter reaction.

If all that fails, the next suggestion is to try optimizing the MgCl2
concentration.  I have not seen this solve a smear problem, only a
total lack of product.

--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
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