HELP: NORTHERN NOVICE

[David C. Logan]

Kim Martin wrote:

>stuff deleted<
>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.

I recommend the low formaldehyde gels described by Fournet et al in 
Focus 10, 5-7 (lost note of year but I can post you a copy if you wish). 
Use thin gels (mine are about 5mm thick which, with the comb I use is 
perfect for a sample volume of 30ul) and run slowly (this is very 
important for nice sharp straight bands). I have blotted by the sponge 
method they dscribe but have recently reverted to the standard 3MM wick 
method (cos I had no sponge) and the results are just as good.
I label probes by random priming using the OLB buffer described in 
Maniatis, purify on Pharmacia nick columns (you could use cheaper 
alternatives) and hybridise (in tubes) with a formaldehyde buffer as 
described in Current Protocols in the section on screening libraries.
Works perfectly.

Good luck,


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david.logan at plants.ox.ac.uk

David C. Logan
Department of Plant Science
University of Oxford
South Parks Road
Oxford
OX1 3RB

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