Home-made Glass Milk

Chris_Upton at darwin.biochem.ualberta.ca Chris_Upton at darwin.biochem.ualberta.ca
Thu Oct 3 21:12:54 EST 1991


He's my method for making Glass Milk.
I've only used it on TAE gels and thought this mix wouldn't work for
TBE gels until some tried it and it worked! (They happened to leave it
melting the DNA for longer than usual).

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  C.


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

Store at  -20  C.



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 C,  mixing frequently until agarose is totally dissolved.
Add 1 microliter 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 microliter 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 microliter) 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 binds 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  ddH2O  (check pH returns to
neutral).
Store final pellet as 50 % slurry in  ddH2O.
Store at  -70  C, working aliquot at 4  C.

 
Chris Upton
Biochemistry
University of Alberta
Edmonton
AB
Canada



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