Big Message Northerns? WT opinion on 2 'funky' protocols

m.champagne at cellbio.duke.edu m.champagne at cellbio.duke.edu
Wed Sep 23 13:57:19 EST 1998


Hi,

the gene i work on has a several huge transcipts (13 and 18 kb), and
several labs have experienced a lot of difficulty getting any signal for those
transcripts.  I heard about two variants of the good ol' Northern protocols,
and wondered if anyone has had any success with those (or knows someone who
has/hasn't).

1-UV crosslinking a hot probe to the RNA mix, then run on a gel, then expose.

By incorporating 4-thio-T into a DNA oligo, then tailing it with radioactive
nucs, I heard you can crosslink it to your RNA, then run the whole thing on a
gel.  No washes need to be done.  The only ref i have about this is RNA 
2:611-621.

2-Drying up the gel and probe the gel directly.

Since big messages are hard to get out of the gel and onto the membrane, i
wondered if it wouldn't make sense to keep the rna into the gel, and probe
the dried up gel itself.  I read a 'paper' which claims they got better 
detection that way (cf Biotechnique 8 (2):162-4, 1990).

Thanks for you help.

m.champagne at cellbio.duke.edu



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