CsCl purification of plasmid DNA

Sebastian Bunka i102pc1 at edvznovell2.vu-wien.ac.at
Tue Dec 28 04:56:42 EST 1993


.>In article <2dgb1o$4tp at canopus.cc.umanitoba.ca> jrichmn at ccu.umanitoba.ca 
.(Joy Richman) writes:>>Dear Netters,
.>>
.>>I have been having an intermittant problem with my Maxi preps. We are
.>>purifying plasmid DNA on a CsCl/ EtBr gradient in an ultracentrifuge,
.>>pulling the bands and then extracting the EtBr with TE saturated
.>>sec-butanol. Unfortunately we cannot seem to find our DNA after
.>>precipitation with 2 volumes of 100% EtOH. The DNA does not seem to want
.>>to come out of solution. We have tried adding 1/10th volume of 3 M NaOAc
.>>(pH 5.2) but no success. Can anyone suggest a reliable extraction
.>>procedure that will result in DNA pellets at the end of the prep?

I use the following procedure:
remove band from tube, fill in PP tube, extract 3-5 times w/ TE-NaCl-saturated 
2-propanol (there must be about 20-25% SOLID NaCl - use the UPPER (Propanol) 
layer !! (somebody told me you can use isoamylalcohol directly - never tried)
add 3-4 !! Vol. H2O (dilute salts!!),
add 2 Vol. 100% EtOH, incubate 2-12h at -20 degC, spin in Sorvall for 30 min 
at 8000 rpm, dry not completely (pellets about 2-5 mm diam.), redissolve in 
100-250 mikroliter H2O.

Cheers
SWB

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