Problem Blotting of Pulsed Field Gels

J. Pat Martinez martinez at puccini.CRL.umn.edu
Tue Dec 17 11:01:39 EST 1996


Hi,

We are currently having difficulty blotting Soybean  high mol. weight
DNA.  In the past, about 1 year ago, everything worked fine, its only
recently that we've had problems.  Even the Lambda standards are faint on
the probed filters.  We've tested the following to no avail.

   Depurination in 0.25M HCL for 0,10, or 20 minutes
   Neutral vs. alkaline blotting
   Traditional capillary blotting (24 hrs.) vs. vacuum blotting
   1% 'standard' agarose vs. 1% Fast Lane agarose
  
We use Whatman 3M filter paper and Hybond N+ membranes.  Any suggestions
to improve our blotting of PFGE gels?

Pat M.



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