plaque hybridization

Leonard N. Bloksberg bloksber at pilot.msu.edu
Thu Nov 4 14:47:00 EST 1993


In Article <9311041740.AA03694 at hamlet.ucdavis.edu> "fzbritt at HAMLET.UCDAVIS.EDU" says:
> A technical question- we're probing a genomic library in lambda with
> an Arabidopsis cDNA.  Can I cram a bunch of those large plaque lift
> membranes into a single 2-inch hybridization tube (used with a rotisserie
> oven)?  It doesn't seem possible that the hybridization mix can coat all
> that tightly rolled surface.  If anyone has had a positive experience
> with this particular technique, let me know.
> thanks,
> Anne Britt
> 
Hi Anne
	When I screened my lambda library, I stuffed all (up to 30) filters
into a single tuperware box.  It worked fine.
	When some companies started offering plastic film to lay against 
your blots (to insure only a capillary layer of hybe buffer) I lost all fear
of stacking membranes.  Their logic was that higher concentration of probe
gave better hybe.  You can increase concentration by either increasing probe
conc. (very expensive) or decreasing volume.  Never bought the system, but
copied it by dramatically reducing my hybe volumes and stacking 1-5 membranes
in a bag or roller bottle.  Works fine for me.  Never tried a whole library
in a single bottle, but I don't see a problem.  The trick is to keep hybe
volume proportional to membrane surface area.  I usually use 50-100 ul per
square cm of membrane.  Friends who try to stick to a given volume per
roller bottle, regardless of membranes inside, have failed miserably.
	Hope this helps.
		Leonard N. Bloksberg



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