Chemiluminescent Southern Blots

Bob Steinberg rsteinbe at etowah.uokhsc.edu
Wed Mar 12 19:08:16 EST 1997


We are interested in using biotinylated probes with a chemiluminescent
detection system for Southern blot analysis of cleavage at methylation
sites in mammalian genomic DNA. We have to look at relatively small
fragments (266-bp down to about 72-bp) and, in the past, have used
32P-labeled DNA probes after blotting digestion products from 30
micrograms of DNA to immobilon-N membranes. We got nice patterns, but
had to do rather long exposures (10-15 days with screens) to get them.
We're now thinking of using biotinylated RNA probes and the Pierce
"SuperSignal" substrate with avidin-HRP for detection. If possible, we
would also like to be able to reduce the amount of DNA required for each
determination.

Does anyone have advice/protocols for probe labeling for highest
sensitivity? Experience comparing RNA with DNA probes (in the past we've
had background problems with 32P-labeled RNA probes)? Experience with
the sensitivity of the Pierce reagent system for Southern blot
detection?

Any help/suggestions would be appreciated.



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