cup@bones.biochem.ualberta.ca (02/24/91)
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.carms@acsu.buffalo.edu (robert m straubinger) (02/28/91)
just a safety note regarding making your own glassmilk -- the finely powdered glass can be quite irritating to skin, and perhaps not the sort of thing one would want to inhale. the colleague who related these concerns to me mentioned that he recycles the Bio 101 glass, rather than making his own. RM Straubinger Pharmaceutics SUNY Buffalo rms@acsu.buffalo.edu