Q: solublility of CsCl in EtOH and iPropOH? (recovery of plasmid

Chris Boyd chrisb at hgu.mrc.ac.uk
Wed Mar 26 05:18:58 EST 1997


Wolfgang Schechinger (wgschech at med.uni-tuebingen.de) wrote:

: Probbly I had made a mistake in the gradient step of my maxipreps. (I 
: got no distinct band, but a EtBr color gradient, decreasing from top) 
: Now I want to recover the plasmids. The soluition contains 
: 1mg/ml EtBr, 1g/ml cesium chloride, all in TE buffer (10 ml overall). 
: I'm thinking of a precipitation w/ ethanol or isopropanol. Will I 
: prec out the cesium chloride, too? What could be the reason of the 
: observed phenomenon?

The caesium chloride will come out of solution unless you dilute. What
people normally do after a CsCl prep is to extract the EtBr, add two
volumes (in your case it would be 20 ml) of water to the prep and then
ethanol precipitate (no need to add salt, two volumes as usual, in your
case 60 ml). I don't know what will happen if you don't extract the
EtBr first. It will probably co-precipitate with the DNA. Frankly, I
think you'd be better off repeating the prep from scratch.  Your EtBr
conc seems about right, but your CsCl conc is a bit vague.  The
critical thing is for the final density of the solution in the tube to
be around 1.55. If your recipe does not lead to solutions of this
density, it is wrong.  If you exceed 1.55 by too much, the risk is that
the CsCl will crystallize capriciously during the run and lead to a
dangerous and very expensive rotor failure.  As a precaution against
this, always do CsCl spins at ambient temperature (say 22 degrees).
Some people have an obsession with never letting plasmid DNA get hotter
than 4 degrees. Nothing untoward will happen to purified plasmid if
kept at ambient temperature for a few hours, especially if it's in CsCl
and EtBr.  In fact by far the best way of storing plasmid long term
(decades) is in gradient conditions, in a sealed tube protected from
the light at room temperature.  Of course even I'm not old enough to
have verified this directly...

Best wishes,
--
Chris Boyd                       | from, | MRC Human Genetics Unit
chrisb at hgu.mrc.ac.uk             |  not  |  Western General Hospital
http://www.hgu.mrc.ac.uk/~chrisb |   for |   Edinburgh EH4 2XU, SCOTLAND



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