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From: cup@bones.biochem.ualberta.ca
Newsgroups: bionet.molbio.methds-reagnts
Subject: Glass milk again
Message-ID: <1991Feb24.014110.20292@cs.UAlberta.CA>
Date: 24 Feb 91 01:41:10 GMT
Sender: news@cs.UAlberta.CA
Reply-To: usercu11@mts.ucs.ualberta.ca
Organization: University of Alberta
Lines: 83

Sorry about my first post of the recipe, but the symbol for "micro"
was deleted.
Also note:
1) this doesn't work with TBE gels (so they tell me)
2) mix the glass slurry well (vortex+++) before use.
3) if various mixes when isolating DNA are vortexed too
   vigorously, then DNA will shear to some exent.
  
Here's the recipes again,
 
PURIFICATION OF DNA BY BINDING TO GLASS  POWDER

Binding and Wash Solutions

NaI solution:        90.8 gm  NaI
                             1.5 gm Na2SO4

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


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

Store at  -20 C.

DNA Purification:  easiest in 1.5 ml microfuge tubes.

To purify DNA from agarose gel, weigh gel slice. 
Add  2 - 3 ml NaI solution per gram of gel.
Incubate at  37-50 C,  mixing frequently until agarose is 
totally dissolved.
Add 1 microlitre  of glass slurry per  microgram  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 microlitre 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 microlitre) and elute DNA at  
50 C  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 gm  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 conc. 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 C, working aliquot at 4 C.

NB This recipe doesn't work for TBE gels
Also mix glass slurry very well before use.

Cost for 2.5 kg of glass powder is approx $3

 
Chris Upton  usercu11@mts.ucs.ualberta.ca


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