Genomic S. blot problems

Stephen R. Lasky Stephen_Lasky at brown.edu
Mon Feb 26 07:26:09 EST 1996


Karen:

Martin's questions are right to the point of your failures because your
washing conditions are, if anything, not stringent enough. For stringent
conditions (for genes homologous to your probe) you can use 0.1X SSC (or
SSPE which is a better buffer), 0.1% SDS (you didn't mention SDS in your
recipes, do you use it), at 60 to 65 degrees for 30 minutes.

Also, are you sure your restriction digests are working, and how much DNA
are you using per digest (you should use at least 10 to 15 micrograms/lane
on the gel).

Finally, as Martin pointed out It's always nice to run a positive control.

SRLasky

-- 
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Stephen R. Lasky Ph.D.   Brown U/Roger Williams Medical Center,  Providence, RI.   
Phone: 401-456-5672     Fax: 401-456-6569     e:mail: Stephen_Lasky at brown.edu
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America may be unique in being a country which has leapt from barbarism to decadence without touching civilization.  John O'Hara.
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