Amount of hybridization probe to use

kim at kim at
Thu Mar 12 14:52:23 EST 1992

Hello group:

I would like to know how to figure out how much probe and hybridization mix to
use for a given number of blots.  Rules of thumb on this subject are usually
given in terms of  cpm per area or volume of mix per area.  With the advent of
roller bottle-type hybridization apparatus, the volume of hyb mix is supposed
to be reduced for a given area of blot, but does the amount of probe get
reduced as well?  That is, is the concentration of nucleic acid in the
hybridization or the number of cpm also get reduced?  Also, is the expected
relative amount of target on the blot an important consideration?  I am
planning to do plaque-lifts on an ordered array of plaques on nitrocellulose. 
The amount of cpm per area is suggested in Maniatis for a ss cDNA probe with a
given suggested pfu/area.  If I halve the pfu/area, do I halve the amount of
probe as well?

I was once told by another grad student that it is not necessary to have blots
swimming in hybridization mix.  It is sufficient to have them well wetted. 
Does this make any sense to more experienced investigators?

Sorry for so many open-ended questions.  thanks for your replies.

Daniel Kim

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