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 
am
>confident that the target desired exists in abundant quantities.
>
>Does anyone out there have any ideas?  I have played around 
unsuccessfully
>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.

good luck

kent



More information about the Methods mailing list