CsCl purification of plasmid DNA

Paul N Hengen pnh at fcs260c2.ncifcrf.gov
Wed Dec 29 14:39:13 EST 1993


In article <i102pc1.87.2D2002DA at edvznovell2.vu-wien.ac.at>
i102pc1 at edvznovell2.vu-wien.ac.at (Sebastian Bunka) writes:

>> 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.

I extract the removed band 3-5 times with isoamyl alcohol until the pink color
is gone. Then dialyze several hours against 0.5-1.0 liter of TE buffer. After
removing from the dialysis bag, I add 1/5 vol of 10 M ammonium acetate and mix
by pipetting. Slowly add 2.5 volumes of cold (-20 C) 95% ethanol to cover the
top of the DNA soln. Gently begin mixing the two phases by swirling. The DNA
will form a visible clot of precipitate within 5 minutes. Spin, remove EtOH,
dry the pellet, and resuspend in H20.

*******************************************************************************
* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
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