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From: cup@bones.biochem.ualberta.ca
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: GLASS_BEAD_PURIFICATION_DNA
Message-ID: <1991Feb21.204616.17568@cs.UAlberta.CA>
Date: 21 Feb 91 20:46:16 GMT
References: <009447D1.0CC2B140@wystan.bsd.uchicago.edu> <1991Feb21.094414.6987@mcclb0.med.nyu.edu>
Sender: news@cs.UAlberta.CA
Organization: University of Alberta
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In article <1991Feb21.094414.6987@mcclb0.med.nyu.edu> levyd@mcclb0.med.nyu.edu
writes:
>In article <009447D1.0CC2B140@wystan.bsd.uchicago.edu>,
loren@wystan.bsd.uchicago.edu writes:
>> Can anyone recommend a source of glass beads suitable for use in DNA 
>> purification?  I cannot locate the source cited in the article by B. 
>> Vogelstein. 

I think my last note about glass milk didn't get to the world distribution
of usenet. (just changing over from mail to usenet, I like it already).
 
Well, I've been making my own glass milk for a couple of years, the recipe was
given to me by Paula Traktman at Cornell Med School who got from.....
 
It works great and costs peanuts. So I tend to use it for everything.
Purify ds DNA for sequencing
Switch restriction buffers
purify vector and insert before ligation
 
I'll tag the recipes at the end.
 
PLEASE use the following for E-mail   usercu11@mts.ucs.ualberta.ca
 
Chris Upton

PURIFICATION OF DNA BY BINDING TO GLASS  POWDER

Binding and Wash Solutions

NaI solution:        90.8 g  NaI
                      1.5 g Na2SO4

in  100 ml  H2O. Filter through Whatman No.1. Put dialysis bag containing  0.5
g Na2SO4  in bottle to keep solution saturated. Store foil-wrapped at 4 oC.


NEET Wash:	100 mM  NaCl
	            1 mM  EDTA
	           50 %    EtOH
	           10  mM  Tris pH 7.5

Store at  -20 oC.

DNA Purification:

To purify DNA from agarose gel, weigh gel slice. 
Add  2 - 3 ml NaI solution per gram of gel.
Incubate at  37-50 oC,  mixing frequently until agarose is totally dissolved.
Add 1 5l  of glass slurry per  5g  of DNA.
Incubate on ice 5-10 mins, mixing occasionally.
Spin 5-10 secs in microfuge, remove and discard supernatant.
Wash glass pellet with 250 5l NaI (or 10 x volume of glass if larger).
Spin and wash pellet  2-3 times with  EtOH wash (same volume).
Dry pellet well, removing all residual liquid (air dry or use Kimwipe
carefully).
Resuspend pellet in  H2O  or TE  (> 10 5l) and elute DNA at  50 oC  for 5-10
mins.
Spin 1 min in microfuge and remove eluted DNA in supernatant.

DNA is ready for ligation, restriction, radiolabelling etc.
DNA bind to glass at high salt and low temp, elutes at low salt and high temp.!

(To purify DNA from solution, add 3 volumes of NaI solution, immediately 
add glass and put on ice).

PREPARATION OF GLASS POWDER

Use silica 325 mesh  (a powdered flint glass available from ceramic shops)

Resuspend 400 g  of glass powder in  800 ml  ddH2O  in a  2 litre flask. 
Stir for 60 mins.
Allow to settle for  90 mins.
Take the SUPERNATANT (which contains the "fines" of interest) and pellet in
Sorvall 
(GSA rotor, 10 mins at 6000 rpm).
Resuspend pellet in  200-300 ml ddH2O.
Add nitric acid to 50 %.
Bring close to boil in fume hood.
Allow to cool.
Pellet glass as before, wash pellet 4-6 times with JddH2O  (check pH returns to
neutral).
Store final pellet as 50 % slurry in  ddH2O.
Store at  -80 oC, working aliquot at 4 oC.

Cost for 2.5 kg of glass powder is approx $3


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