Northern trouble!

Richard V. Giles giles at liv.ac.uk
Tue Aug 25 08:01:45 EST 1998



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:
>

Me too

>
> (1) Did you boil your total RNA to denature it and eliminate secondary
> structure before loading it onto the gel?

Don't do this! You will hydrolyse your RNA resulting in greatly reduced
signal.

Assuming that you are using formaldehyde - agarose gel electrphoresis,
resuspend your RNA in formamide using half the volume that you wish to load.
Heat to ~65C for 5 to 10 mins and vortex. Add 1 volume of a 2X conc MOPS /
formaldehyde / dye buffer. Repeat the heat / vortex cycle.

Namjin,

If I understood your post correctly, you had a generalised high background
with "holes" in the background where the rRNA and markers were.

Suggestions

1. Include yeast tRNA in the hybridization buffer (~100ug/ml)

2. Use an amphipathic nylon membrane (eg Hybond N, Nytran etc)

These two should reduced non - specific binding of your probe to the membrane.
With sufficient non - specific binding the concentration of probe drops to
such an extent that hybridization to your target sequence is v. inefficient
and no signal is obtained.

Good luck
--
Richard V. Giles Ph.D.
School of Biological Sciences
University of Liverpool
Tel +44/151 794 4389
Fax +44/151 794 4349
http://www.liv.ac.uk/~giles/





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