CsCl purification of plasmid DNA

Jim Owens jow at helix.nih.gov
Wed Dec 1 09:21:01 EST 1993


In article <2dgb1o$4tp at canopus.cc.umanitoba.ca> Joy Richman,
jrichmn at ccu.umanitoba.ca 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 had pretty much stopped using CsCl when I last was preparing plasmids. 
Here is the method I used:

(This portion is a slight variation of Birnboim in Methods in Enzymology
vol 100, p 243)
SolnI: 0.9g glucose, 1.25ml 1M TrisHCl pH8, 1ml 0.5M EDTA pH8 to 100ml.
SolnII: 8ml 5M NaOH, 10ml 20% SDS and 182ml H2O.
SolnIII: 29.9g KOAc, 5ml 90% formic acid to 100ml.
Transfer each culture to a 1 liter bottle and spin at 4! for 15min in J6B
rotor at 4200rpm. (Use of lysozyme seems unnecessary for most commonly
used vectors)  Resuspend each cell pellet in 10ml SolutionI and transfer
suspensions to 45Ti Oak Ridge tubes.  Let stand 15min at room
temperature.    Add 20ml SolutionII to each tube.  Mix until suspension
clears. Let stand on ice for 10 min.  Added 15ml ice cold
SolutionIII(6/26/89).  Mixed as gently as possible and put on ice for
15min.
Spin in 45Ti rotor for 15min at 30,000rpm at 4!.  Transferred
supernatants to 150ml Corex bottles.  Added 0.6 vol iPrOH (~26.3ml).  Let
stand at room temperature for 15min.  Then spin at 8Krpm for 45 min in
GSA rotor at 20!.  Rinse pellets with 95% EtOH.  Let air dry. 
 
(This part was modified from Ze!ev Lev, Anal Biochem 160:332-336(1987))
Dissolve each pellet in 2ml TE.  Transfer to 1.5ml microfuge tubes,
~0.6ml per tube.  Add to each tube 0.6ml of 10M LiCl to precipitate large
RNAs and polysaccharides. Spin in microfuge for 10 minutes.  Recover
supernatants and divided them between two SW40 tubes.  Precipitate
plasmid DNA with 2 volumes of EtOH.  Spin down in SW40 rotor at 30Krpm
for 15 minutes at 15!.  Pellets are tight enough to pour off supernates. 
Redissolve each pellet in 0.6ml TE and transfer to microfuge tubes.  
Spin for 30 seconds to pellet insoluble material.  Layer the top 0.5ml or
so of supernate over 4.3ml 3M NaCl, 0.1mM EDTA, 20mM TrisHCl, pH8, in
SW55 tubes. Spin at 50,000rpm, 20! for 90 minutes.  (Plasmids are
pelleted; small RNAs stay in solution.)  Carefully pour off supernatants.
 (Save supernates in case the plasmid has not completely pelleted
although it has never been necessary to use the supernates.)  Redissolve
pelleted plasmids in 0.4ml TE.  Transfer to 1.5ml microfuge tubes, mix in
8!l 5M NaCl.  Precipitate DNAs with 0.8ml EtOH.  Rinse precipitates with
70% EtOH and dry.  Dissolve precipitates in an appropriate volume of TE.


This can be accomplished in one day from overnight, amplified cultures. 
I would only use CsCl now if I needed the centrifugation time to do other
things.

Good luck,

Jim Owens



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