SMEARS WITH PCR

robin carper RCARPER%JHUHYG.BITNET at JHUVM.HCF.JHU.EDU
Wed May 18 15:13:40 EST 1994


Someone posted that, when using Barnes' PCR protocol, he was getting nothing
but long smears in his wells.  These are probably the result of BSA in the
buffer and can be removed by heating the samples for five minutes at 95 prior
to adding the polymerase mixture.  Using gelatin in place of the BSA might also
remove the smears.
    I got the smears and then tried DNase treating the PCR samples, phenol/
 chloroform treating, EtOH precipitation of samples, and Quiagen PCR purif-
 ication kit treatment.  Only the DNase and phenol treatments removed the
smears.  Puzzling until I treated the sample with DNase buffer alone (37
degrees for three hours) and found it removed the smear. Heating prior
to the rxn removed the smears from my next run.  So...I think it is some
strange salt/BSA interaction that stains.  I also found that increasing amounts
of 2N Tris base (2 microliters, 5 microliters, 10 microliters) diminished the
smear.  Go figure. The BSA, I think, does need to be solubilized somehow, so
you might also want to try 0.1% Triton X 100 (used in Vent and Pfu buffers), if
you don't want to "hot start."
                      Good luck



More information about the Methods mailing list