help needed with southern
leach at bu.edu
Fri Aug 4 11:32:37 EST 1995
HOw are you doing the hyb...in a bottle or a bag...
amount of prehyb doesnt really matter....for me..
for the hyb i always keep it 1million cpm per ml of fluid (go up to 5 mill/ml)
in bottle hyb...large bottles...about 20ml...small bottles about 10ml
...letting us know what method you are using will help..
PAUL CANNNISTRARO (pcannist at opal.tufts.edu) wrote:
: I am working with genomic DNA (10ug per lane of gel) and I am somewhat at a
: loss in figuring out the appropriate amount of hybridization fluid to use.
: Miniatis recommends .2ml of pre-hyb per sq. cm of nylon membrane, which comes
: out to 34ml for 14x12cm. However, the amount of hyb solution is not specified,
: but one could infer that it is the same, since it is suggested to replace the
: pre-hyb with an identical solution. The controversy arises when I take into
: account that Maniatis recommends 10-20ng/ ml of labeled probe during
: hybridization and 100ng of probe altogether. Thus, I should have 5-10ml of
: hybridization solution?
..... Martin Leach Email:leach at bu.edu
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