stripping Northerns

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Wed Oct 29 17:23:16 EST 1997


At 13:42 10/29/97 -0800, Dianne Emslie wrote:
>
>I am having problems stripping Northern blots probed with DIG labelled
>oligos and hope someone may have a better method.  I have tried boiling in
>0.1% SDS which seems to remove the chemiluminescent substrate but not the
>oligo and I have tried the DIG Northern probe-stripping solution which
>contains 50% fomamide. That seemed to strip the probe off but also the
>RNA.  Does anyone have any suggestions? I would be most grateful for your
>help.

Dianne:

I've used this with success for both northerns and southerns.  I orginally
obtained it from the Bios Laboratories manual for their genomic southerns.

Stripping Protocol:   Strip wash the blot for re-probing or long term
storage.  Bring 250 ml of 0.1 XSSC to a boil.  Remove from heat.  Add 2.5
ml of 10% SDS (1.25mls of 20%).  Float blots in solution (do not pour strip
washing solution over blots) and shake at room temperature for 15 minutes.
Repeat.  Monitor probe removal by Geiger counter and exposure to X-ray
film.  Store air dried blot at 4'C in a sealed bag.

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"
 http://www.uth.tmc.edu/`dhavilan 
 Voice: 713.500.2413  FAX: 713.500.2424
"A conclusion is simply the place where you got tired of thinking."
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