Dig detection for Southerns.

Klaus Salger salger at wap18.zi.biologie.uni-muenchen.de
Mon Feb 27 16:32:27 EST 1995


Paul N Hengen (pnh at fcs260c2.ncifcrf.gov) wrote:
<snip>
: I'm using the Southern-Light [TM] kit from Tropix which also uses the CSPD
: substrate for alkaline phosphatase. For detection, I dilute the stock CSPD
: 1:100 in 0.1 M Diethanolamine, 1 mM MgCl2 pH 10.0. I only use 10 ml at a
: time and then throw it out, but your posting implies I can save it to reuse.
: How do I store this for using it again say, one or two weeks later? Is it
: okay to put it at 4C? Isn't it now contaminated with alkaline phosphatase
: that possibly came off the membrane...and is now in the process of being
: phosphatased to bits?

Paul,
maybe you're interested to try a different procedure. My procedure is almost
exactly as described in Boehringer's "DIG System User's Guide for Filter
Hybridization".
Put a 200ul drop of staining solution (same as yours) on a sheet of plastic,
add the blot, add second film, gently wipe out airbubbles, incubate some
minutes, wipe out staining solution and expose. This can be conveniently
done in a plastic bag with 2 of the 3 closed sides cut open. I find it simple
and cheap and one doesn't have to care about contaminations or decay of
the CSPD.

Cheers
  Klaus

--
Klaus Salger                phone : ++49 (0)89 5902 -502
Zoologisches Institut       FAX   :                 -450
AG MacWilliams              e-mail: salger at zi.biologie.uni-muenchen.de
Luisenstr. 14
80333 Muenchen
Germany



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