Low stringency Southern trouble
t.barnett at path.utas.edu.au
Thu Feb 15 19:55:21 EST 1996
In article <1996Feb14.150838 at opal.tufts.edu>, kmorris1 at opal.tufts.edu (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!
I guess the next thing to do is to keep lowering the hybridisation temperature until you do get some hybridisation. I have successfully done low stringency Southerns using the DIG kit from BM. The hybridisation buffer is 5xSSC, 1x blocking reagent (I don't know what is in this), 0.5% sarcosine and 0.1% SDS. I usually start at a low hybridisation temperature, say 37C and keep rising the temperature until I get some degree of specificity.
Tim Barnett fax: 61-02-354833
Department of Pathology phone: 61-02-354825
University of Tasmania email: t.barnett at path.utas.edu.au
Hobart, Tasmania 7000
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