NOTES ON THE PURIFICATION OF DNA WITH GLASS MILK

The following notes are ed ited from a variety of comments downloaded from "bionet.molbio.methds-reagnts" bulletin board on isolating DNA from agarose gels using the "Glass Milk" technique published by Patterson, PNAS 76:615 (1979). We have not made our own glass milk, so I can not attest to their accuracy.

Special thanks to those who provided the majority of these comments:

  • Chris Upton (Chris_Upton@darwin.biochem.ualberta.ca)
  • Don Back, Department of Biochemistry, Queen's University, Kingston, Ontario, CANADA (BACKD@QUCDN.QueensU.CA)
  • wenyen (wyk238@csd4.csd.uwm.edu)
Procedure:
  1. Weigh the gel slice in a 1.5 ml microfuge tubes. Add 3 volumes of NaI solution per gram of gel. Incubate at 37-50°C, mixing frequently u ntil the agarose is totally dissolved.
  2. Mix the glass slurry well and add 1 µl of the glass slurry per microgram of DNA. Incubate on ice for 5-10 minutes, mixing occasionally. Spin 12,000 xg for 5-10 seconds in a microcentrifuge. Discard the supernatant. (The supernatant may be saved to avoid losing DNA).
  3. Wash the glass pellet with 250 µl of NaI solution (or 10-times the volume of the glass pellet, if it is larger). Spin and wash the pellet 2-3 times with the same volu me of ethanol. Dry the pellet well, removing all residual liquid. This may be done using air, carefully wiping with a Kimwipe, or a vacuum drying apparatus).
  4. Resuspend the pellet in at least 10 µl of H2O or TE and elute the DNA at 50°C for 5-10 minutes. Centrifuge for 1 min at 12,000 xg in microcentrifuge and collect the eluted DNA in the supernatant.
  5. is now ready for ligation, restriction enzyme digestion, radiolabelli ng, etc. DNA binds to the glass at high salt and low temperature, and elutes at low salt and high temperature.
Miscellaneous Notes:
  • TBE gels:  Attempts to use glass beads to isolate DNA fragments from TBE gels give variable results. Millipore has developed an immobilized glass containing filter that they use to isolate DNA via the NaI method. They claim it works well with TBE gels if you add sodium phosphate, pH 6.5 to 100 mM final concentration. Apparently, the TBE problem is overcome at lower pH. The composition of BIO-101's TBE modifier is unknown, but you may be able to substitute a less expensive and readily available PO4 buffer.
  • Small DNA Fragments:   A BIO 101 sales representative at the ASCB San Diego meeting explained that the reason glass milk does not bind to small DNA fragment (say, < 500 bp) too well is because of the pH. Lower the pH to isolate these.
  • Note: A 10% sodium sulfite solution can also be used to remove the yellow s taining that results from spills of the NaI.
RECIPES

Preparation of Glass Powder
Use a powdered (not fumed) silica from Sigma, Silica 325 mesh for making glass beads from Cutter Ceramics, 11908 Old Baltimore Pike, Beltsville, MD 20705; 301-595-0720 (@ 50 cents/pound plus $4.00 shipping and handling), or a powdered flint glass available from ceramic shops (2.5 kg @ about $3).
  1. Use silica 325 mesh. Resuspend 400 g of glass powder in 800 ml of double-distilled H2O in a 2 L flask. Stir for 60 minutes. Allow particles to settle for 90 minutes.
  2. Take the SUPERNATANT (which contains the "fines" of interest) and centrifuge at 6000 xg for 10 minutes to pellet the glass (6000 rpm in a Sorvall GSA rotor). Resuspend the pellet in 200-300 ml double-distilled H2O.
  3. Add concentrated nitric acid to 50%. Bring close to boil in a fume hood. Allow to cool. Pellet the glass as above, wash 4-6 times with double-distilled water. The pH should return to neutral. Store final pellet as 50% slurry in double-distilled H2O. Store stock at - 80°C; store working solutions at 4°C.
Binding and Washing Solution Recipes:
    NaI Solution:
         Dissolve 90.8 g NaI
                        1.5 g Na2SO3 (sulfite, not sulfate)
         in 100 ml of H2O. Filter through Whatman No.1 filter paper.
   & nbsp;     Put a dialysis bag containing 0.5 g Na2SO4 in a bottle to keep solution saturated.
        Store foil-wrapped at 4°C.
        NaI solution notes:   Use a saturated solution at RT which is approximately a one pound bottle of NaI in 250 ml of TE. Add one gram o f sodium sulfite. This solution dissolves gels faster and results in a higher binding rate. Store at room temperature. Wash the glass in a 60:40 dilution of this in water (stored at -20°C). Vortex the glass slurry well before use. Some batches of NaI need to be buffered. Once when the DNA was lost, the NaI solution was pH 10! Buffer the NaI solution with TE. We also wash simply with 100% ethanol (i.e., bind in saturated NaI, wash once in 60% saturated NaI, wash once in 100% ethanol, spin dry, elute in 20 ul TE).

    NEET Washing Solution: (Store at -20°C)
        100 mM NaCl
            1 mM EDTA
        50% EtOH
          10 mM Tris-HCl, pH 7.5
    Send comments an d updates to  Dr. Bart Frank, Arthritis and Immunology Program, OMRF
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Proper citation for data acquired from this document is: "Frank, M. B. Notes on the Purification of DNA with Glass Milk. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/glasmilk.html). 1997. Oklahoma City. Revision Date: October 2, 1997."