Non-radioactive probes for Northerns

User Name Mail.Address at ualberta.ca
Thu Nov 23 12:10:24 EST 1995


In article <48vv2h$t9b at dartvax.dartmouth.edu>, Deborah.B.Pedersen at dartmouth.edu (Deborah B. Pedersen) says:
>
>Due to attempts to minimize the amount of radioactive waste in our lab
>(and department) we would like to start to use one of the
>non-radioactive kits for labeling probes for Northerns. We have heard
>that the biggest drawback to these are background problems as opposed
>to limits of dectection. Is this true? I would like to get some
>feedback from others who have tried this and would greatly appreciate
>any advice.  Thank you!

I have labeled cRNA probes with Digoxigenin-11-UTP
 (from Boehringer Mannheim).  Using these probes to probe Northern blots, 
I found that I get a considerable amount of background binding to the 
28S and 18S ribosomal bands and sometimes these are the only two bands
that light up.  
My recommendation is to carefully consider the membrane type, the procedure
used to X-link your RNA to your membrane, prehyb. and blocking solution 
(during detection) incubation times.  As well, isolation of the mRNA from 
total RNA before running a gel is probably a good idea to avoid the above 
mentioned problem
Use my email address if you want to chat more
Karim (ksayani at gpu.srv.ualberta.ca)
 
>
>Deborah B. Pedersen
>Dartmouth Medical School
>Hanover, NH 03755 
>e-mail Deborah.B.Pedersen at Dartmouth.edu



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