Low stringency Southern trouble

Richard Daines rdaines at q.continuum.net
Thu Feb 15 10:52:49 EST 1996


KELLY THOME wrote:
> 
> I'm hoping someone can help me with remedial Southern analysis technique :-).
> I'mtrying, without much success, to do low stringency Southerns (ie, hunting for
> family members in one species/looking for homologs in other species).  My hyb
> and washing conditions for high stringency blotting are pretty standard, I
> guess... hyb is 6X SSC/10X Dextran Sulfate/ 0.5% SDS/ 50micrograms/ml ssDNA at
> 65 degrees, washing is 1X SSC/0.5% SDS at 65 degrees.  I tried lowering the
> SDS to 0.1% and hybing at 42, but then I got NO signal, not even the signal I
> see at high stringency.  I'm getting sort of frustrated at blank blots and
> would appreciate advice from someone with experience in this sort of thing.
> Thanks in advance!
>                 -Kelly
> 
> Kelly Thome                     kmorris1 at opal.tufts.edu
> Department of Pathology
> Brigham and Women's Hospital
> Boston, MA
>         All thoughts expressed here are my own!

Go to 2x salt (or higher) and decrease your temp to 55C.  I don't think 
changing the SDS conc. will help that much.
-- 
Richard J. Daines
Mystic, CT

rdaines at q.continuum.net



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