Northern blot problem(s)

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Wed Mar 17 21:20:50 EST 1999


In article <36F01CC5.C4F7B077 at agora.ulaval.ca>, Tahar aboulkassim
<abh346 at agora.ulaval.ca> wrote:

> 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.
> Thank you
> M. Taha Aboulkassim

[Newsgroup list heavily pruned]

Can you trim your probe down?  In some circumstances
I've found that a 1.5 kb probe gave a messy signal
but a 0.3 kb subclone was much cleaner.  There were
also some old postings to the effect that some
vectors have homology to rRNA and hybridise tightly
so you need to trim away any vector fragments.

If you can't see your specific signal at all I'd
strongly suggest performing polyA selection or
switching to RNase protection analysis.
     Good luck,
          Bernard
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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