help needed with southern

Dimitri Papadopoulos papa at comp.bioz.unibas.ch
Thu Aug 10 14:55:16 EST 1995


In article <1995Aug4.111250 at opal.tufts.edu>, pcannist at opal.tufts.edu (PAUL
CANNNISTRARO) 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?
The amount of Buffer does not matter so much, but I usually worl with as
little buffer as possible. For a bottle (HYBAID) I usually use 15ml, maybe
20ml if
I have more than 2 membranes and meshes per bottle.
Just make sure that you take off all prehyb solution beore adding your hyb
solution, or else you will find your buffer volume doubled (meshes can
retain an awful lot of buffer)
Check your bottles after 15' for bubbles or places the buffer doesn't reach.



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