ribosomal RNA and northerns

John S Jacobs Anderson jacobs at azstarnet.com
Mon Sep 14 23:35:59 EST 1998


In article <8396847 at dasher.Dartmouth.EDU>,
Christopher.M.Hammell at Dartmouth.EDU (Christopher M. Hammell) wrote:

>Hello,
>    I have a mRNA that runs with the same mobility as 28S rRNA. The
>ribosomal RNA causes my message to run awkwardly and results in a
>smeary banding patterns when I probe it for northerns.  I don't want to
>poly A+ select because in my mutant strains, polyadenylation is
>inefficient at the non-permissive temperature.

This is just a thought -- I'm not responsible if it doesn't work! 8^)=

Could you get an oligo that hyb's to the 28S rRNA and use RNaseH digestion
to cleave it? The only problem I can think of is that there's a boatload
of 28S (relative to most mRNAs)...

Duh!(hand slaps forehead)

Cut your message of choice! Order an oligo that's complementary to your
message and treat your mRNAs with RNase H. Make sure that you get products
that are distinguishable in size (ie, position the oligo asymmetrically in
the mRNA). You'll have to use 2 oligo probes or a random primed probe in
your Northern,so as to see both products, but it should work.

(If you need a protocol for the RNase H digestion, I think there's one on
the Parker Lab web site at <http://www.mcb.arizona.edu/Parker/>. If not,
email jacobsan at u.arizona.edu and I'll hook you up.)

john.
(from home.)
-------------------------------------------------------------
John S. Jacobs Anderson      http://www.azstarnet.com/~jacobs 
  Apprentice Geek                        jacobs at azstarnet.com    
   Journeyman Molecular Biologist             Tucson, AZ, USA





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