Hi R. Aviner >Hello, >I recently encountered a problem working with [32]P labelled DNA probes for >RNA detection. > >I pre-hybridize positively charged nylon membrane in 7% SDS 0.3M Na2PO4 for >several hours, then add my labelled probe (purified using a PROBER column) >for overnight incubation, followed by washing in 5% SDS 20mM Na2PO4. > >This used to work fine for my purposes, but now I repeatedly get very hot >membranes and the radioactivity simply won't go away, even after extensive >washing (and stripping under harsh conditions). I assume this may be due to >non-specific binding of labelled nucleotides, but I fail to understand why. >Any ideas?"
I usually have salmon sperm DNA and BSA to block the membrane in my pre-hybridization buffer, or else it seems that you would get high background, nothing prevents your probe from sticking to the membrane (like your RNA did). Jonathan Perreault
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