Help: Blotting DNA from acrylamide gel

Stuart Brown SBROWN at GAES.GRIFFIN.PEACHNET.EDU
Tue Jul 18 08:41:06 EST 1995


This is a real stumper for me.  I need to make a Southern blot of some
PCR products after separating them by size in a polyacrylamide gel.  I
need to use acrylamide in order to resolve small size differences between
products (2bp SSR alleles) and I need to probe in order to verify that
my PCR products really contain the repeats that I'm supposed to be
amplfying.  According to "Current Protocols" you can't use simple
capillary transfer on polyacrylamide, they reccomend electroblotting.
I found an electroblotter (Pharmacia "Multiphor II") and gave it a try
using my "best guess" conditions (0.5X TBE buffer, 200 mA, 20 min) with
absolutely no useful result (ie. blank film after probing filter and
long exposure). I called the Pharmacia tech help line and was told that
they don't have a reccomended protocol for blotting DNA with their machine
because nothing that they have tried worked very well.  Come on, this has
got to be possible!
 
Does anyone out there have any experience blotting DNA out of acrylamide?
No reasonable suggestions sneered at.
 
Cheers - Stuart Brown
 
Stuart Brown                       |   Plant Genetic Resources
                                   |   Georgia Experiment Station
INTERNET:                          |   1109 Experiment Street
  SBROWN at GAES.GRIFFIN.PEACHNET.EDU |   Griffin, Georgia 30223-1797 USA
BITNET:   SBROWN at GRIFFIN           |   Fax: 404-229-3323



More information about the Methods mailing list