high background with dig in situ

sticknbd sticknbd at miranda.cc.vanderbilt.edu
Wed Jun 16 21:29:22 EST 1993

I have high background with dig in situs I'm doing
right now.  At Boehringer Mannheim's (BM) suggestion,
I let the first colour reaction go three days, and got
a lot of background.  I let the second set go one hour
and I still had a lot.  I tried serially diluting the probe,
and each slide still had background,  including both
sense and antisense.  BM recommends 10ng/ul probe.
I've made minour stringency adjustment (5C higher
for hybridization) with no difference.  I did notice
the first time, where I was checking prot K efficiency,
that the shorter the prot K digest, the darker the back-
ground.  I still figure it's the colour rxn, though, so I'm
going to watch it from time zero and try to catch it
right away, as soon as positivity (which is always
present in the right places) develops, and prior to
background development.  I didn't have this problem
with tritium.   Anybody have any suggestions, comments?
sticknbd at mirand.cc.vanderbilt.edu

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