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RNA dot blot protocol?

RNA dot blot protocol? - Protocols and Methods Forum

RNA dot blot protocol? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 12-28-2006, 04:52 PM
Teresa Bori-Sanz
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Default RNA dot blot protocol?



Hi Dr. Klaus Eimert,
I was wondering if somebody got back to you. I am also trying to do a DOT
BLOT but I would like to try it to detect tRNA. If so, could you let me know
what was the best protocol?

Best wishes,
Teresa


Hi evreybody,

I am looking for a fast and "fool-proof" RNA dot blot protocol.
I want to do some ISH on paraffin sections using DIG-labeled probes. To
make sure that my probes are OK and specific, I thought to do a RNA dot
blot for control.
I thought to just spot the total RNA on the membrane and let it dry (no
manifold or such). The protocols I found in the books and on the web differ
quite a bit, though. In some, RNA is spotted on the dry membrane, some
prewet with water or SSC (2x to 20x). Also, some don`t denature the RNA at
all, some "boil" (65-70°C) and some use NaOH.
So, does anybody care to share her/his good/bad experience with a certain
protocol? Any traps I should to avoid?
Thanks in advance!

Regards,

Klaus



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