help needed with southern

[martin LEACH]

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..

Martin

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?


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.....          Martin Leach                Email:leach at bu.edu 
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