David L. Haviland, Ph.D.
HAVILAND at KIDS.WUSTL.EDU
Thu Sep 1 14:33:39 EST 1994
In <Pine.3.87.9408291250.D1876-0100000 at csuvax1> obrien at CSUVAX1.MURDOCH.EDU.AU writes:
> Dear Netters
> We have been trying unsuccessfully to do northern blots of fungal RNA.
> the RNA should be rRNA. However we see nothing. I would like to ask
> people who have experience of northerns, what is the most appropiate
> transfer buffer, and membrane to use. Grateful for any suggestions.
I'd like to stand on my personal soapbox. I would wholeheartedly recommend
Virca et al (1990) BioTechniques, 8:370-371. I normally run 20-25 ug per
well. It uses sodium phosphate as a transfer buffer rather than 10XSSC and
is transferred to Hybond N+. The next day you will see 18 & 28S RNA on the
N+ and even with a UV transilluminator I have hardly seen any residual RNA
in the agarose gel. Transfer is quick! I've done this in as little as 14
hours - I have yet to try a vacuum blotter though. In contrast, we have
folks who go with 10XSSC but often let the transfer go for 48 hours.
End of soapbox...
Hope this helps,
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