Alkaline gels-Better method?

[David T Wong]

I'm trying to separate PCR products on 4% Alkaline agarose gels with NaOH 
and EDTA.  If I run them overnight the bands are diffuse.  If I run them 
over 5 hours (400mA and about 90V in BioRad wide minisub cells) with lots 
of buffer cooling, I get uneven running.  The top of the gel with cool 
buffer runs faster than the bottom on the tray.  I have to take pictures 
at a 45 degree angle.  Urea gels don't work for me.

Does anybody have a better way of running alkaline gels at reasonable 
field strengths.  Is there another high pH buffer system that is 
compatable with agarose or acrylamide (acrylamide hydrolyzes in the 
NaOH/EDTA buffer).  Is there a more stable gel?  I read about AAEE 
acrylamide but where can I buy it?  Any suggestions would be appreciated.


David Wong
Division of Gastroenterology
Stanford University
wong at leland.stanford.edu