Northern blot problem(s)

Tahar aboulkassim abh346 at agora.ulaval.ca
Wed Mar 17 16:18:16 EST 1999


Hi, There,

I have always a problem when I do my northern blot experience. The
problem is the non-specific hybridization of my probes  with 28S and 18S
and I have a strong signal for these RNAs. furthermore, the specific
signal is either weak or absent. I have changed many parameters in order
to improve or to resolve this problem. But, always  have the same
problem.
It is suspected that the blocking agents may be the source of the
problem, I then used other blocking agents like poly A+, with those used
previously e.g. Salmon sperm DNA (Gibco), and Denhardt solution. We used
several different probes ( DNA and RNA probes) and conditions of
prehybridization/hybridization : 60°C for RNA probe and 42° for DNA
probe (with formamide), but the result remained the same.

I would be grateful if someone can give me an idea ( may be someone had
already the same problem) of what could be the problem.

Thank you

M. Taha Aboulkassim
Onco-urological laboratory
Cancer Research Center
Laval university
Quebec (Canada)
E-mail : abh346 at agora.ulaval.ca




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