NORTHERN BLOTS

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.  

Some Omitted...

> 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.

Phil:

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,
David




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