in situ with DIG-riboprobe

[Klaus Salger]

CV Laboratory (system at cvlab.harvard.edu) wrote:
: Hello.

: I have been doing in situ hybridization on carotid arteries using
: digoxigenin labelled cDNA.   For some reason, I always get non-specific
: binding with sense probe.   When Northern was done to test the antisense
: and sense probes,  sense gave strong 18S and 28S band when antisense probe
: just gave the expected band.   I raised the temperature at which I
: hybridize and wash, but I did not have much luck.    The same thing
: happens to another gene which I am interested in.  Does anybody have any
: idea why I have such problem, and/or how to solve it?

: Thank you very very much.
: --SK

I assume that you used riboprobes. If so, pBluescripts MCS might be
the problem. The sequence between the XbaI- and SacI-site hybridizes to
rRNAs, as far as I remember. This problem was reported earlier in this
newsgroup, so you might find additional information when searching the
old articles (on gopher://net.bio.net).

Hope this helps
  Klaus

--
Klaus Salger                phone : ++49 (0)89 5902 -502
Zoologisches Institut       FAX   :                 -450
AG MacWilliams              e-mail: salger at zi.biologie.uni-muenchen.de
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