Blocking nonspecifics on Southerns-your choice

James S. Sutcliffe jamess at mbcr.bcm.tmc.edu
Sat Nov 20 09:12:35 EST 1993


Victor,

When you say the salmon sperm DNA was degraded, what does that mean? I don't think you
can effectively block membranes with nucleotides. Typically people use "sheared" or
sonicated salmon sperm DNA. What I do to prepare sheared blocking DNA is to drop a known
weight of the dry DNA (e.g. from Sigma) into a flask with a volume of TE (10, 1) to
produce a 10 mg/ml concentration when dissolved. The flask with undissolved DNA is
autoclaved for 20 min, which both dissolves and shears the DNA. This is then used both
in prehybridization and hybridization at an approximate concentration of 250 ug/ml. I
denature the salmon sperm DNA prior to addition to the buffer, but this is probably
somewhat redundant as the autoclave step denatures the DNA. Regardless, blocking DNA ust
be denatured. Ours ranges in size from 200-1000 bp.

If you're seeing "spots" on your membrane, that's caused by unincorporated 32P. You need
to do something (more?) to purify your probes from the unicorporated 32P (e.g. G50
sephadex columns). A general background (not spots; not restricted to lanes) IS
generally a blocking problem, in my experience.

The buffer I like to use, may not help you much if you're trying to save money, but it
works great!!

0.25 M NaPhosphate
0.25 M NaCl
1 mM EDTA
10% PEG-8000
7% SDS
1% BSA

The BSA presumably works as a blocking agent also, in this buffer. This buffer is
somewhat tricky to make, but it's, by far, the best buffer I've evr used. If anyone
wants details I could post them.  BTW, I don't think SDS acts to "block" membranes.
					       ^^^^^
Hope this helps.

Jim Sutcliffe
Institute for Molecular Genetics
Baylor College of Medicine



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