In situ hyb with Digoxigenin
Dr. Kent L. Nastiuk
nastiuk at rockvax.rockefeller.edu
Tue Feb 6 13:14:20 EST 1996
In article <4f71vi$20dg at pulp.ucs.ualberta.ca>,
ksayani at gpu.srv.ualberta.ca (Karim Sayani) wrote:
>I have previously conducted in situ hybridization using riboprobes
>labeled via in vitro transcription with Digoxigenin-UTP as the label.
>However, upon switching to a new probe, both sense and antisense probes
>give me positive staining. I looking in a cell culture system where I
>confident that the target desired exists in abundant quantities.
>Does anyone out there have any ideas? I have played around
>with the stringency of the washes. I either get the staining with both
>probes or it all disappears.
first, while it is important to wash stringently, it is equally
important to eliminate non-specific binding in the first place. have
you tried raising your hybridization stringency in conjuction with
increasing the wash stringency?
second, you might also try RNAse treatment during the washes.
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