@#! smears on PCR ???

Ann M. Yezerski ayezersk at moose.uvm.edu
Tue Sep 27 16:56:38 EST 1994


Frederick Garbrecht (FRED at bmt.mcw.edu) wrote:
: I am going crazy with smears on my PCR gels.  Using the same set of 
: primers, sometimes I get very nice product band(s), but sometimes I 
: get a diffuse smear from top to bottom with no bands.  Any hints as 
: to what may be going on to cause this?  Thanks--

If all of the solutions and programmed steps are the same for every reaction:
It seems as if the PCR sometimes work and sometimes doesn't.  The smear, 
if only faint, would just be your DNA from you sample.  One of the 
problems could be not vortexing your Master Mix if you use one, and 
therefore, not dispersing the Taq evenly to all of your samples.  Or, 
it's possible that the Taq was not added at all.  Make sure that you see 
the Taq go into the reaction as a stream of glycerol.  Sometimes, with 
such small amounts, it simply does not get added.  




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