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problems with probe binding to 28S and 18S RNA only on Northerns

problems with probe binding to 28S and 18S RNA only on Northerns - Protocols and Methods Forum

problems with probe binding to 28S and 18S RNA only on Northerns - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 09-11-2006, 02:30 AM
Wendy Ingram
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Default problems with probe binding to 28S and 18S RNA only on Northerns



Hi Sasha,

Occasionally over the years I have also had the ribosomal bands on
northerns light up brightly with a probe. It has generally been from one
of 3 causes, all of which are easily fixed.

1. A common cause is tiny traces of contaminating DNA/cDNA in probe
preparations. For example if you directly amplify a probe by PCR using
random-primed cDNA as the template, then use this product as your probe
rRNA binding often occurs. Tiny tiny traces of ribosomal DNA sequences
can cause a major problem (as there is generally many orders of
magnitude more rRNA on your blots than RNA for the gene of interest).
Sometimes even gel purifiying does not remove the problem completely.
However, after cloning the probe of interest into a t-tailed vector (eg
GemT-easy) and then re-amplifiying, probes can be "cleansed" of any
residual template DNA and then behave beautifully.

2. If you are making your probes using any electrophoresis steps then
the rRNA binding problem can occur if the tank has not been cleaned
beforehand. When you cut out bands traces of DNA from previous users are
always floating about the tank, and if you are unlucky a titch of
something that binds to rRNA will will end up in your probe... Sometimes
all it takes to turn a rRNA binding probe into a beautiful clean probe
is making it again with a nice clean tank. Nowdays I always give tanks a
good clean with SDS and hot water before making probes.

(It's interesting how easily DNA traces end up probes from other things
in a tank - when I use to do lots of Southerns we use to run a ladder on
one side of our probe isolation gels, and our probe digest/PCR on the
other - we would always end up with the ladder on our Southerns lighting
up when using our "gene specific" probes isolated from the other side of
the gel... It was actually really useful, so we use to do it deliberately)

3. On occasion some probe sequences light up the ribosomal bands no
mater how carefully you make the probe. Sometimes it is faint and the
transcript of interest is away from the 18 and 28S, othertimes there is
an enourmous amount of binding and it stuffs up what you want to see. I
guess this happens for me with about 1 in 20 UTR gene probes I make. In
these cases I simply pick another suitable region of the UTR, or a novel
part of the coding region, and design another probe. This always fixes
the problem.

Good luck,
Wendy Ingram.




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Wendy Ingram, PhD
Senior Research Officer
RCH Cancer Research Laboratory
Phone: +61 7 3636-9211
Fax: +61 7 3365-5455
Email: initial.name at uq.edu.au

Postal & Delivery Address:
Level 3, Foundation Building
Department of Paediatrics and Child Health
The University of Queensland
Royal Children's Hospital
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Herston, QLD 4029, Australia

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18s , 28s , binding , northerns , probe , problems , rna


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