northern stripping

lappel at eagle.wesleyan.edu lappel at eagle.wesleyan.edu
Fri Nov 19 13:20:24 EST 1993


I did compare, when doing a lot of reprobing of Northerns and Southerns during
a walk.  Southerns strip fine by the cheaper method (3 cycles of pour on some
boiling H2O with a little SSPE and SDS, let sit 10-20 minutes, pour off), but
Northerns required the stronger & more expensive method (50% formamide, 10 mM
NaPO4, pH7 for 1 hr at 65C, then rinse 15 min in 2xSSC, 0.1% SDS room temp to
remove the formamide).
	I was able to re-use the filters at 6 times each, often more.
	
	Laurel F. Appel     		LAPPEL at EAGLE.WESLEYAN.EDU
	Wesleyan University
	Middletown, CT 06459 

                                                              
> In article <2cfamm$b6c at mserv1.dl.ac.uk>, suter at VAX.mpiz-koeln.mpg.d400.de writes:
> +With regard to Southerns we (and the FBI) have found that stripping in 55%
> +formamide, 2xSSPE, 1% SDS at 65*C for 60-90 minutes results in the cleanest
> +removal of probe and the least loss of target (i.e. can reprobe MANY times
> +e.g. 8-10 times). 
> 
> before everybody starts using your probably truly succesful method, did you ever
> compare this method within one experiment to stripping with e.g. TE/
> boiling for one hour ? I usually chunk my filter in either 1 % SDS with some 
> TE and heat to 80 for 1 hour, or, in the case I am dealing with a 
> riboprobe, I just add a spot of RNAseA to TE, and heat to 80 for one hour. 
> I probe most filters more than 10- 15 times, no problem. 
> 
> If you can use a simple buffer.... do !
> 
> clemens
> 
> ===============================================================================
> Dr. Clemens Suter-Crazzolara, PhD
> Max-Planck-Institut fuer Zuechtungsforschung
> Abteilung Genetische Grundlagen der Zuechtungsforschung
> Carl-von-Linne Weg 10,        D-50829 Koeln, Germany
> Tel.xx49.221.5062-221    Fax.-213      e-mail: suter at vax.mpiz-koeln.mpg.d400.de
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