northern blotting

Sam Gray Samuel.~remove-this~Gray at nottingham.ac.uk
Fri Feb 28 09:35:16 EST 1997


On 20 Feb 1997 00:45:58 -0800, daemon at net.bio.net
ad.mccormick at ulst.ac.uk ("a.mccormick") wrote:

>Hi, Does anyone have a fool proof (if that exists in molecular biology) 
>northern blotting method using total RNA. All my attempts so far have 
>ended in disaster. 

>thanks,

>a.mccormick

Hello.
I think you're right in doubting the existence of a fool proof
protocol - that's the philosphers stone of mol.biol.! What probes have
you tried using? I've had numerous attempts using oligo's ranging in
size from 20 to 50bp and with various different labelling methods, all
with very little success. By far the most successful probes in my
experience are riboprobes transcribed off of linearised plasmids (I
use the Promega RiboProbe kit with alpha-32P CTP) but there are
problems with this too. To be honest with you I've given up using
total RNA and now use mRNA almost exclusively. There are lots of
poly-A RNA extraction kits available, and while expensive, if you do a
rough cost-analysis then it usually works out cheaper to do one blot
with mRNA that works than ten blots with total RNA that don't work!
If you want to email me with some more details of where you think your
blots are going wrong I'll be happy to swap ideas with you.

Samuel Gray



____________________________
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samuel.gray at dial.pipex.com
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