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

Dom Spinella dspinella at chugaibio.com
Tue Mar 25 17:48:58 EST 1997


Wolfgan Schechinger wrote:
"Hi all.
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?

Any suggestions are welcome!"

Wolfgang:
Well it seems to me that there are at least 3 possible reasons for your
failure to observe a plasmid band in your maxi-prep.  First, its
possible that there was no DNA in the solution to which you added CsCl
and EtBr.  Did you get good bacterial lysis?  Did you see a pellet when
you precipitated the plasmid from the cleared lysate?  Second, it
possible that you have mis-weighed your CsCl.  You can check to make
sure that the refractive index of your solution is 1.395-1.400.  Lastly,
it is possible that you didn't spin the tubes long enough.  You don't
say what kind of rotor, centrifuge speed, or spin time you used.

Anyway, if you try to alcohol precipitate the DNA (assuming it's in
there) from the CsCl solution you will very likely bring down the CsCl. 
You might try dialyzing the solution for a few hours in several changes
of buffer before precipitation.  Alternatively, you could precipitate
the whole thing, and then remove the CsCl by successive washes in 70%
ethanol (but there's likely to be a LOT of CsCl in that pellet!). 
However, absent some trivial error, I would bet that there was no
plasmid DNA in there to begin with!  Hope this helps.
D. G. Spinella



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