plaque hybridization

[Brian Smith-White]

In Article <19931104154734.bloksber at thomashaw-at.css.msu.edu> "bloksber at pilot.msu.edu  (Leonard N. Bloksberg)" says:
> 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
> 
	An interesting response to the question. Anne, if you wish to 
hybridize a single membrane which has either i) its shortest dimension that 
is greater than the circumference of the hybridization cylinder or ii) its 
largest dimension is greater than the height of the cylinder, is there a 
problem? If you can successfully accomplish a hybridization with a single 
location of membrane-atop-membrane, than there is no barrier to multiple 
locations of membrane-atop-membrane. I would not suggest that this allows you
to pack the hybridization tube with membranes, but you could reasonably 
expect to introduce enough to generate 5 to 10 layers. You could also pack 
the hybridization tube with more than one of these concentric piles if the 
height of the cylinder allows. To parphrase Leonard, unless you are 
going to invent gas-phase hybridization, you have to adjust the volume of 
hybridization buffer accordingly. However, you have to have more fluid than 
that required to merely wet the membranes. The notion that a capillary layer 
of hybridization buffer {with probe at a particular specific activity (and 
implicitly at a particular concentration of polynucleotide)} will be more 
effective than an ocean of the same hybridization buffer defies physics. 
Annealing of probe to target is related to the concentration of the probe and 
target, not the volume in which the physics is occurring. I am still in the
dark ages and do not use the roller bottle/rotisserie instruments. I prewet
the membranes in a solution composed of everything in the hybridization 
buffer except the radioactive probe. I introduce the wet membranes into bags
and then provide the desired amount of probe in a small volume of the 
solution used to prewet the membranes. In principle this method can be 
applied to the roller bottle/rotisserie equipment. The desired amount of
probe is determined by the number of membranes in the enclosure. For 132 mm
diameter membranes I use 5,000,000 to 10,000,000 precipitable counts per
membrane.
	Illegitum non carborundum.
	Brian (the dinosaur graduate student) Smith-Whi