In Article <4929u0$77o at pulp.ucs.ualberta.ca>, Mail.Address at ualberta.ca (User
>In article <48vv2h$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.
Interesting. I've done the same and have not had this problem. A good cRNA
probe should not hybridize to any significant extent to ribosomal RNAs. If
it does, I'd investigate the probe specificity (it's easy to compare the
probe to the rRNA sequence), and hybridization/washing conditions (time,
temp, salt, etc.). In my experience, dig-cRNA probes work very well. I have
noticed significantly lower sensitivity when compared to 32P.
Thomas T. Aquilla, Ph.D. .***. .***. .***. .***.
Molecular Physiology and Biophysics * | | | * * | | | * | | | *
University of Vermont Medical College* * | | | * * | | | * * | | | *
Health Science Complex, Given E-201* * | | | * | | | * * | | | *
Burlington, VT 05405-0068 '***' '***' '***' '***'