dig labelled Riboprobe whole mount insitu?

P. Jones/ P. Scotting mbzpj at unicorn.nott.ac.uk
Wed Oct 12 06:31:10 EST 1994

In article <doerther-0710941246040001 at mac165.hogan.nwu.edu>,
doerther at nwu.edu (Dan Oerther) wrote:

>    My specific questions are for those who have had experience with Mouse
> embryos (Not to ignore the advice of Drosophilia experts, but in this case
> the tissues may react rather differently - but please offer any advice!!).
>    1) are my hybridization conditions too stringent?
>    2) what do you use to remove excess/nonspecific riboprobe i.e. washes?
>    3) what levels of Proteinase K treatment do you recommend?
>    4) any other advice - besides Give Up, this method will never work.
>We carry out such in situs on chick embryos, but I know our methods are similar to others working in mice.

			We use very similar stringency to you, ie 70C hyb and 65C washes,
similar buffers. We do not hydrolize 1-1.5 Kb probes and we do not post
treat with RNase. Proteinase K is 10ug/ml but prewarmed to 37C and then
15min on the bench.
My guess is that not hydrolizing might help and otherwise we have found
that titrating probe concentration can make a big difference.

Paul Scoting and Maria Rex
Dr. Paul J. Scotting
Dept. of Biochemistry,
University of Nottingham,
Queen's Medical Centre,

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