help needed with southern

PAUL CANNNISTRARO pcannist at opal.tufts.edu
Fri Aug 4 11:12:50 EST 1995


   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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