HELP: NORTHERN NOVICE

Kimberly Martin 100131.655 at CompuServe.COM
Sat Jun 1 12:41:46 EST 1996


Dear All,

I have recently been awarded a small equipment grant to purchase 
a u.v. crosslinker and a northern blotter.  Having now purchased 
the equipment, I feel somewhat obliged to use it!  I intend to 
look for specific splice variants of the gene in question.  I 
have the technology but, sadly, not the expertise.  In the past, 
I have successfully performed in situ hybridisation on paraffin 
sections and can sometimes get PCR to work, so I feel fairly 
confident that, in time, I can master the art of Northern 
hybridisation.  What I would like from this group is any and all 
advice, wisdom, warnings, you would care to offer, from probe 
design/labelling to running the gel, blotting, hybridising, etc.

In my Northern blotting fantasies, I perform asymmetric PCR using 
nested primers around the exons of interest, incorporating 1/4 
dUTP:dTTP to produce the probe and incubating the blot with an 
anti-dig antibody conjugated to alkaline phosphatase and finally 
visualising with BCIP/NBT (this worked a treat with the ISHH, 
except someone else produced the probe).  Is this (a) the long 
way around the barn?  (b) feasible? and/or (c) sufficiently 
sensitive?  I want to avoid radioactivity at all costs.  If the 
above scenario is impractical, I am willing to try, for instance, 
ECL.  Any advice, anecdotes, etc. is welcome.
Kim Martin, Dept. of Neuropathology, Inst. of Psychiatry, London



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