Removal of CsCl from CsCl-EtBr gradient preps

Conrad Halton Halling chh9 at quads.uchicago.edu
Mon Sep 9 11:53:00 EST 1991


In article <0094E602.8B8D6680 at aclcb.purdue.edu> 
muriana at aclcb.purdue.edu (murianap) writes:
>I am posting to inquire if anyone can give me a easy way of getting rid of
>the CsCl remaining in EtBr-extracted CsCl chromosomal preps?  I have 
>routinely used Amicon Centricons for plasmid preps, but believe these will 
>get clogged if I use them for chromosomal preps.  I've tried the method in 
>the new Maniatis (add 3 vol d.H20 + 2 vol EtOH) but that didn't work.  Is 
>there any other method besides dialysis?
>
>Thanks in advance,
>Peter M. Muriana
>    Name: murianap
>Internet: muriana at aclcb.purdue.edu
>   Phone: 317-494-8284

I think the description in the "new Maniatis" (Sambrook et al., 1989.  
Molecular Cloning, 2nd ed., p. 1.46) is just plain wrong.  Here is what
I routinely use for plasmid preps -- it should work for chromosomal preps too:

     1)  Measure the volume of the DNA + CsCl solution.
     2)  Add two original volumes of TE or dH2O and mix.
     3)  Add six original volumes of 95% ethanol and mix.

The DNA will precipitate at room temperature.  I spin at 12,000 x g for
15 minutes to recover the DNA.

Example:  For 1 ml of DNA + CsCl solution, add 2 ml of TE and mix, then
add 6 ml of 95% ethanol and mix.


-- 
Con Halling
chh9 at midway.uchicago.edu



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