Desalting of Plasmid DNA after Cesium Preps
chrisb at hgu.mrc.ac.uk
Thu Jul 9 07:05:05 EST 1998
Obaid Yusuf Khan (9321531k at clinmed.gla.ac.uk) wrote:
: Does anyone know about a method to remove Cesium from the plasmid DNA
: after a Cesium Chloride-Ethidium Bromide density centrifugation. I have been
: using dialysis for desalting but its time comsuming. I have also used
: columns, but they can only take upto 500 mcg of DNA and you usually
: get over a
: mg of DNA in cesium preps. I have also used ethanol ppt method, but I tend to
: loose a lot of DNA this way.
: I will appreciate any help/tips for desalting the plasmid DNA, which consumes
: less time and does not damage the supercoiled structure of DNA.
Dialysis may be fiddly and time-consuming, but I think it's the best
way to get maximum yield of pure, high-quality DNA. How much time do
you use? You can, if desperate, get away with 2 x 2 hour dialyses, though
it's best to play safe and do 1 x 2-5 hours, 1 x overnight. In any
case, always do at least one change of buffer -- some people seem to
think leaving DNA to dialyse in one batch of buffer in a HUGE beaker is
OK, *as long as you leave it long enough*! They probably also think a
mole is only a small furry animal. To convince yourself something is
happening (and fast) you can see the salt streaming out of the dialysis
tubing the second you drop it into the beaker -- just look at it
against a bright background.
I think you always lose a lot doing ethanol precipitation, particularly
in the washing stage which is necessary to remove salt traces.
You have to make a choice: time or yield + quality.
Chris Boyd | from, but not \ MRC Human Genetics Unit,
Christopher.Boyd at hgu.mrc.ac.uk | on behalf of / Western General Hospital,
http://www.hgu.mrc.ac.uk/Users/Christopher.Boyd \ Edinburgh, EH4 2XU, SCOTLAND
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