Materials

1. Glass beads: These can be purchased or prepared in the laboratory as described below. The preparation involves boiling nitric acid so take full safety precautions. Mix 250 mL of powdered glass flint available from glass supply companies (e.g., Eagle Ceramics Inc., 12267 Wilkins Avenue, Rockville, MD 20852) with 500 mL sterile water. Allow the heavier particles to settle over 1 h. Decant the fines into centrifugation tubes, and spin for 5 min at 1000g. Resuspend the pelleted fines in 200 mL sterile water. Add 200 mL of concentrated nitric acid, and bring to boil. Carry out this step in an efficient fume hood. Allow to cool and then centrifuge (1000g, 5 min) to recover the glass beads. Discard the nitric acid carefully. Wash several times in distilled water (i.e., cycles of resuspension and spinning) until the pH of the suspension is neutral. Store the glass bead slurry as a 50% (w/v) slurry at -20°C, working stocks at 4°C.

2. Gel electrophoresis equipment.

3. 50X TAE buffer stock: 2.0 M Tris base, 1.0 M sodium acetate, 0.05 M EDTA.

4. Ethidium bromide (10 mg/mL) in sterile water.

5. Long-wave (365 nM) UV light transilluminator.

6.6 M Nal: Dissolve 90.8 g of Nal and 1.5 g Na2SO3 in water to a final volume of 100 mL. Filter through a 0.45-|im Nalgene filter or Whatman no. 1 paper. Add 0.5 g Na2SO3 to saturate. The Na2SO3 crystals do not dissolve, but serve to prevent the oxidation of the Nal. Store at 4°C protected from light.

7. Ethanol wash solution: 50% (v/v) ethanol, 0.1 MNaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA. Store at -20°C.

8. Sterile (autoclaved) filtered (0.2 |im) Microinjection TE (MITE): 10 mM Tris-HCl, pH 7.4, and 0.2 mM EDTA.

9. Sephadex G50 slurry: Prepared by swelling G-50 powder in water followed by autoclaving.

10. 0.45-|im Millipore filter attached to a 1-mL disposable syringes.

11. Baked glass wool: Bake at 250°C for at least 3 h to destroy all contaminating nucleases.

10.3. Method

1. Digest the DNA with the appropriate restriction enzymes according to the recommendations of the manufacturer.

2. Fractionate the digested DNA in an agarose gel prepared in 1X TAE buffer containing 0.5 |g/mL ethidium bromide.

3. Visualize the DNA on the long-wave UV light transilluminator. Use a fresh scalpel blade to excise the fragment of interest carefully in as small a volume as possible. Transfer the excised fragment to a preweighed Eppendorf tube.

4. Add as much NaI as possible, the minimum ratio being 3:1 (v:w).

5. Incubate at 55-65°C with occasional agitation until the agarose has completely dissolving.

6. Add 1-|L of glass beads (fully dissolved and mixed) for every 2 |g of DNA. Mix well, and incubate on ice for 45 min inverting every 10-15 min.

7. Pellet the glass milk by centrifugation (pulse to 13,600g over and release). Discard the supernatant.

8. Wash with 500 ||L of NaI twice more, spinning briefly (1-2 s) each time to recover the glass milk.

9. Wash with 500 | L of ethanol wash twice.

10. After discarding the supernatant, vortex the glass milk in the small volume of ethanol that remains and respin. Carefully remove as much of the supernatant as possible, but do not let the pellet dry out.

11. Add more than 50 |L of MITE immediately, and incubate at 55-65°C for 15 min to elute the DNA.

12. Pellet the glass beads by spinning at full speed for 2 min. Transfer the supernatant containing the DNA to a fresh tube. After this point, all tubes and tips must be prerinsed with filtered water before use.

13. Prepare a spun Sephadex G-50 column. Block the end of a 1-mL disposable syringe with glass wool. Fill with Sephadex G-50 until all the excess fluid drains out and the beads are packed. Position the column in a disposable 15-mL plastic conical centrifuge tube, and centrifuge at 4000g for 3 min. Equilibrate this column with 100 mL MITE and spinning as before. Do this five times.

14. Pass the eluted DNA (maximum volume 100 |L) through the equilibrated Sephadex G-50 column, collecting the purified DNA in a fresh 1.5-mL Eppendorf tube (with the lid cut off) placed at the bottom of a fresh 15-mL centrifuge tube.

15. Filter the elute through a small 0.45-|im Millipore filter attached to a 1-mL syringe.

16. Determine the DNA concentration by spectrophotometry at 260 nm or comparative ethidium staining with DNA standards of known concentration.

10.4. Notes

1. Many manipulations of DNA and RNA leave the nucleic acid in a solution containing unwanted salts, nucleotides, or radioactive moieties. In most cases, etha-nol precipitation will not entirely remove these or will add further salts. Sephadex gel-exclusion chromatography is often used to purify DNA fragments. Sephadex is a bead formed, crosslinked dextran. The cross linking is carefully controlled to give beads consisting of a network of holes of uniform size and shape. A molecule passing through a Sephadex column will pass through a volume dependent on the size of the molecule. A molecule too large to enter a pore will pass through the volume of the column not occupied by beads. A molecule small enough to enter the pores will do so, and pass though a much larger volume and take longer to pass through the column. The exclusion limit is the size limit of the molecule that can just enter the pore. Molecules of intermediate size will be fractionated by their molecular weight. Thus, for example, Pharmacia makes G-50 Sephadex gel-filtration matrices that fractionate in the range of 500-10,000 daltons. This-matrix can fractionate a mixture of DNA and salts cleanly, the DNA being excluded from the pores and passing straight through and the salts and nucleic acids being retained within the pores. Additionally, the sample does not become appreciably diluted.

2. Glass bead DNA isolation kits are available (e.g., Geneclean II by Bio 101).

3. The recovery of DNA fragments between 500 and 800 bp by glass bead isolation is relatively low. Also there is a risk that large DNA fragments (>15 kb) may be broken during the wash cycles. If a large DNA fragment binds to two or more beads, then on washing and separation of the beads the DNA strand may be broken.

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