Colony Hybridisation with biotinylated oligo
browns at ccu.umanitoba.ca
Fri Jan 22 16:14:00 EST 1993
In article <1993Jan20.145525.17865 at gserv1.dl.ac.uk> ahill at crc.ac.uk (Mr. A.F. Hill) writes:
>I'm trying to set up a system for doing colony hybridisations using a
>biotinylated oligo (23-mer) and using streptavidin-biotin-alkaline phosphatase
>and NBT/BCIP to get the colour reaction...
>I've spotted the oligo onto a filter and can read a signal down to 10pg but
>when i hybe with the oligo and develop the colour i get a nice purple
Sounds to me like you need better blocking. Some kits come with
a "Secret Blocking Reagent" which is probably BSA. Also, you can try
pre-hybridizing with a mixture of salmon sperm and plasmid without insert.
Colony blotting is a very dirty proceedure. I've also read of a technique
where a library is subdivided by plating transformed bacteria on a large
number of plates (maybe 1000 to 10,000 clones per plate), then the colonies
from each plate are pooled for a large scale plasmid prep (keeping a replica,
of course). The plasmid pools are then tested by dot blot hybridization.
Pools with positive signal are sudivided and the proceedure repeated, etc.
Stuart M. Brown If you can remain cool when all
U. of Manitoba, Dept. Plant Science Around you are in panic,
Winnipeg, Manitoba, CANADA
browns at ccu.umanitoba.ca Then you surely misunderstand the situation
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