Northern trouble!

Christie Malazdrewich malaz002 at gold.tc.umn.edu
Mon Aug 17 17:47:57 EST 1998


In article
<Pine.SOL.3.91.980817092403.21938B-100000 at godzilla3.acpub.duke.edu>,
Namjin  Chung <nc1 at acpub.duke.edu> wrote:

<When I exposed my 
<membrane, after hybridization, to phosphorimager and x-ray film, I saw 
<generally high background and two ribosomal RNA bands as well as RNA size 
<markers being white.  However, I could not detect any of the RNA that I 
<wanted to see.  What would be wrong with this?

Hi Namjin,

I sympathize with your situation - I, too, was long a student in the
"Teach Yourself Northerns" school. Now things are working fine. A few
questions for starters:

(1) Did you boil your total RNA to denature it and eliminate secondary
structure before loading it onto the gel?
(2) Is it possible that the sequences of interest are not present? That
is, did you run a positive control sample known to contain mRNA for the
gene you're looking for?
(3) What system are you using for detection? Is it possible that your
problem lies in faulty labelling rather than absence of target or lack of
hybridization?
(4) To what stringency are you washing your blots after hybridization?

Cheers,
Christie.

Christie Malazdrewich
Clinical and Population Sciences
University of Minnesota
malaz002 at gold.tc.umn.edu



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