Northern trouble!

ORAC ORACII at aol.com
Tue Aug 18 11:48:28 EST 1998


In article <malaz002-1708981747570001 at x245-51.cvm.umn.edu>,
malaz002 at gold.tc.umn.edu (Christie Malazdrewich) wrote:

>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?


Good questions. Here are a couple more:

(5) Did you purify your probe with a Sephadex G-50 spin column?
Unincorporated nucleotide can give very high background?
(6) What hybridization buffer are you using? Personally I have had much
better results using Church buffer rather than Denhardt's solution.

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