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

Bernard Murray bernard at elsie.nci.nih.gov
Tue Mar 25 17:22:16 EST 1997


In article <5h923i$fn8 at mserv1.dl.ac.uk>, wgschech at med.uni-tuebingen.de says...
>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,
	If you think it really is a setup problem rather than just a
lousy yield I'd try adding more ethidium and/or checking the density
and respinning the preparations.  I know it takes another night to
see the result but it is fairly painless to do at this stage.
	If your sample had a lot of residual protein this could soak
up the ethidium and stop you from seeing the band - the top of the
gardient shouldn't be too pale.
	You can rescue DNA from the CsCl solution by precipitation with
isopropanol (I even added ammonium acetate to ensure that all the DNA
came down).  This will result in a *huge* pellet but you can gradually
wash this away with several washes with 70% ethanol and spinning.  If
you are starting with big volumes of CsCl/DNA (>5 ml) this will take a
loooooong time and so dialysis against TE would probably be far quicker.
Maybe gel filtration would help...
	There's an outside chance that the DNA in the CsCl sample will
bind to diatomaceous earth or silica gel (eg. Wizard prep resin) as
binding is normally done in a high salt buffer.  You could try this
with a small aliquot (bind - wash - elute with TE) to see if it will
work - please post/e-mail me if it does!  You may have to reduce the pH
to eg. 5 for binding to work best.
	You will eventually have to remove the ethidium bromide so
extraction with water-saturated butanol would probably be the best way.
	Good luck
		Bernard

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)




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