probe stripping

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Fri Jun 4 09:02:07 EST 1999


At 05:14 6/4/99 -0700, Halina Sobolewska wrote:
>Can someone suggest ways of stripping oligo probes from dried nylon
>membranes. I have tried boiling them in water containg 0.1% SDS. Should
>membranes be kept wet and if so, why?

Yes.  I came across a wonderful method in the Clontech manual that, in my
hands, works very well for both Southerns/Northerns and has worked the few
times I've tried with oligo probes.

1) To a metal pan, add 250 mls of 0.1X SSC and bring to a roaring boil.
2) Turn off heat and immediately add 1.25 mls of 20% SDS.
3) Immediately add blot and submerge it.  
4) Place on a rocker platform and rock  10-15 minutes.  <--"basting"
5) When cool or warm, check with G_counter and and repeat if necessary.  
6) Image o/n (film or imager) to determine level of previous probe.
7) Re-probe!

With this method, I've often been able to re-probe genomic southerns and
northerns 3-4 times before having to "retire" the blot.

In your case, with the blot already dried.  You are likely stuck until it
decays out.  There is something about dried blots wherein the probe is
locked into place and I've never been able to both remove the probe from a
dried blot and still have the blot in good enough shape to be re-probed.
I've always found those mutually exclusive and often easier just to re-run
the blot.  

Suggestion: After I'm finished exposing a blot, I always place it at 4'C
still wrapped in saran wrap.  I've placed one of those plastic magazine
holders in the cold room and just "pack-rat" all my blots in the holders.
I'd advise folk just to keep them wrapped and cold.

Hope this helps,
David
=============================
 David L. Haviland, Ph.D.
 Asst. Prof. Immunology 
 University of Texas - Houston, H.S.C.
 Institute of Molecular Medicine  
 2121 W. Holcombe Blvd.  
 Houston, TX  77030 
 Internet:"dhavilan at imm2.imm.uth.tmc.edu" 
 Voice: 713.500.2413  FAX: 713.500.2424
 ------------------------------------------------------  
The hardness of butter is directly proportional to the softness
of the bread.
=============================



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