CsCl purification of plasmid DNA

[Sebastian Bunka]

.>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

            `oo'
          ( O  O )
             vv \\
                  \\
		    \\		  email:
                      [ Sebastian.Bunka at vu-wien.ac.at ]
                        phone:                   FAX:
                +43-1-71155260          +43-1-7149110
Location: earth, europe, austria, vienna  Inst. of Bacteriology  Vet.Univ.