Strange Southern Problem

z z-suldan at ski.mskcc.no.spam.org
Thu Dec 4 09:53:12 EST 1997


In article <EKo5o4.K6M at midway.uchicago.edu>, bbettenc at midway.uchicago.edu
(brian bettencourt) wrote:

>         Been doing fly genomic Southerns w/ great success over the past
> year: nytran mb's, neutral transfer, turboblotter (S&S) system, DIG-probe,
> colorimetric detection (I look at hsp70s, very good conservation, super
> strong signal).  Anyway, recently things have gone awry and no one can
> figure it out.  Basically these are the symptoms: ridiculously high
> *generalized* background (not in lanes), but ***only on the top ~1/2 of
> the blot***.  It looks like a shadow of the gel, complete w/ wells, down
> to a line roughly corresponding to where ~1.5Kb frags are.  Below that the
> shadow is gone and the blot looks fine.  Very strange.  Don't think it's
> blocking, Ab, developing, or anything post-hyb b/c the bkgrnd is only on
> part of the blot.  This has been repeated now for three blots.  It's not
> due to overlapping in the hyb tube.  The only things I've changed recently
> are 1) new restriction scheme, smaller frags, thus slightly stronger gel. 
> 2) OVERNIGHT GEL - could this be leading to an EtBr front or something? 
> Any ideas?  No one has heard of this.  Thank you very much.
> 
> 
> -- 
>
_______________________________________________________________________________
>  Brian Bettencourt  *  b-bettencourt at uchicago.edu  *  (773) 702-8096 / 5228
>  Organismal Bio & Anatomy  *  http://http.bsd.uchicago.edu/~feder/brian.html
>
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I've got to think about this one some more... but here're some quick
possibilities.

1. Overlapping. I mean while washing, not while hybing. Try washing in a
large tupperware box or the like.

2. Could it be you film cassette is hot? It's happened in our lab before.
Check it with a geiger or a piece of film o/n. If you're keeping your
blots damp/wet, this could be a very real possibility.

Good luck

Zal



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