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:
> (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
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.
If I understood your post correctly, you had a generalised high background
with "holes" in the background where the rRNA and markers were.
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.
Richard V. Giles Ph.D.
School of Biological Sciences
University of Liverpool
Tel +44/151 794 4389
Fax +44/151 794 4349
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