The following procedure provides a simple method for isolating high molecular weight DNA, suitable for restriction endonuclease digestion, from a nucleus-enriched, low-speed-sedimentable fraction of yeast cells. Because this DNA is prepared from a fraction with greatly reduced level of soluble and high-speed-sedimentable substances, the final DNA preparation is likely to be free of the contaminating substances which frequently inhibit restriction endonucleases and/or otherwise interfere with analysis of DNA prepared from whole yeast cells by conventional techniques. In addition, the use of glass beads to break open whole yeast cells speeds and simplifies this procedure, compared to procedures in which spheroplasts must be prepared.
1. Grow yeast cells under whatever conditions are desired, to whatever concentration is desired. Stationary phase cells break as easily with glass beads as log phase cells.
1a. In the case of S. pombe cells, we recommend adding sodium
azide to a final concentration of 0.1% (from a 10% stock solution) immediately
before harvesting, then rapidly chilling by swirling on ice for about 1
minute. These steps minimize DNA nicking and are especially important for
subsequent neutral-alkaline 2D gel analysis. We have not seen evidence that
these steps are important in the case of S. cerevisiae cells.
2. Harvest cells as desired (typically 5000 rpm, 5-10 min).
3. Wash the cells once or twice with cold water.
4. Resuspend the cells at 1.5 - 2 X 10^9/ml in ice-cold Nuclear Isolation Buffer (NIB). This buffer consists of 17% glycerol, 50 mM MOPS buffer, 150 mM potassium acetate, 2 mM magnesium chloride, 500 µM spermidine, and 150 µM spermine. The pH is adjusted to 7.2 after all ingredients are dissolved.
5. The cell suspension is mixed with an equal volume of glass beads in a round-bottomed or conical-bottomed hard plastic centrifuge tube. We use Arthur H. Thomas Catalog #5663-R50 (0.45-0.52 mm diameter) beads. The largest volume we have successfully used is 13 ml suspension plus 13 ml glass beads in a 50 ml plastic conical-bottomed tube. To homogenize larger amounts of cells, multiple tubes may be used, and once the cells have been adequately homogenized, they may be removed from the beads so that the beads may be used over again with a separate portion of cells. There are devices available commercially which allow shaking of much larger volumes with glass beads, but we have not adequately tested such devices, nor have we tested other types of homogenizers.
6. The following operation is carried out in a cold room. The tube with cells and beads is covered to prevent leakage. The tube is agitated vigorously (at full speed) with a healthy Vortex mixer or similar device for a period of 30 sec. The tube is then placed in an ice bath for at least 30 sec. This operation is repeated as many times as necessary (5-15 in our experience) until more than 90% of the cells have been broken (determined by measuring the ratio of intact cells to cell ghosts by phase contrast microscopy; quality of liberated nuclei may be assessed by fluorescence microscopy using a DNAspecific stain such as DAPI). Breakage is most effective when all the beads are lifted and swirled and when the suspension reaches a height at least three times its normal height as it is swirled around the side of the tube.
7. The supernatant solution is removed from the glass beads with a pipette. In order to recover the significant liquid volume which is trapped in the spaces between the glass beads, the beads are washed twice with 1.5 - 2 volumes (each wash) of NIB.
8. The washes and original supernatant are combined and centifuged (cold) at 8000 rpm in a Beckman JA-20 or Sorvall SS34 rotor for 10 min.
9. Resuspend pellet at a final concentration of 2 X l0^9 cell equivalents/ml in 50 mM Tris, 50 mM EDTA, 100 mM NaCl, pH 8.0.
10. Add 0.05 (1/20) volumes of Sarkosyl NL-30 to the pooled material, to achieve a final Sarkosyl concentration of 1.5%. Mix.
11. Add solid Proteinase K, or concentrated Proteinase K stock solution, to a final concentration of 300 µg/ml.
12. Incubate at 37° for 1 hr, shaking at 15-20 min intervals.
13. Centrifuge (cold) at 5000 rpm (Beckman JA-20 or Sorvall SS-34) for 5 min to pellet cell ghosts. The supernatant should have only minor turbidity.
14. For each ml of supernatant solution, add 1.05 g of CsCl. Encourage the CsCl to dissolve by gentle mixing. Don't mix vigorously or foaming will result. After the CsCl has dissolved, the refractive index may be checked (optional). It should be 1.394.
15. Measure the final volume and then add 0.001 volumes of a 5 mg/ml stock solution in water of Hoechst 33258 dye. Mix.
16. Transfer to ultracentrifuge tubes of appropriate volume.
17. Balance tubes, cap, and centrifuge in an angle rotor at 35,000 rpm and 20° for 2 days, or use a vertical rotor at 40,000 rpm for 16-20 hrs.
18. Identify the nuclear DNA-containing band under long wave (360 nm) UV light. Collect the band.
19. Add 2.5-3 volumes of diluted ethanol (75%) at room temperature. Cap tube with Parafilm and mix by slow, gentle inversion. A fibrous precipitate should form. This precipitate is likely to stick to the tube; that's fine. As the aqueous and ethanol phases mix, the fibrous precipitate will first enlarge as more and more DNA precipitates, and then it will contract as it becomes dehydrated by ethanol. A fine, thin, turbid precipitate is also likely to form. This fine precipitate will be enriched for lowermolecular weight DNA and may possibly contain some RNA.
20. Separate the supernatant and turbid precipitate from the fibrous precipitate. This is easy to do if the fibrous precipitate stuck to the side of the tube: simply pour off the supernatant. Otherwise it may be easier to catch the fibrous precipitate on a clean glass rod or with a pair of clean forceps or with a P-1000 Pipetman and pull it out of the supernatant solution. It will usually not be necessary to centrifuge, and centrifugation should be avoided because it will lead to contamination of the fibrous precipitate with material from the fine, turbid precipitate.
21. Rinse the fibrous precipitate twice or 3X with 70% ethanol, and then allow it to dry briefly (about 10 min) in air.
22. Dissolve the fibrous precipitate in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) to achieve a final concentration of 100 - 500 µg/ml. For example, use 1 ml to dissolve the DNA from 3 X 10^10 yeast cells. Due to the high molecular weight of the DNA, the process of dissolving will take a long time. Try leaving the DNA in TE in the refrigerator or cold room overnight or even as long as two days. On the following day, pipette very gently with a wide pipette to help disperse the DNA. Several hours additional solution time, after the dispersal by pipetting, may be necessary to get the DNA fully into solution.
23. Determine the DNA concentration by measuring OD (at 240, 260, and 280 nm) of a 1:50 dilution in TE. Warning: this measurement gives total nucleic acid concentration. Residual RNA in your prep will also contribute to OD. Therefore it is also essential to check the quality and quantity of the DNA prep by conventional gel electrophoresis of a small aliquot (usually 1 µl) followed by ethidium bromide staining. At the same time, include a DNA standard of known concentration in your agarose gel. Virtually all the DNA should be found in a single highmolecularweight band, except for sharp bands due to plasmid DNAs. Comparison of the fluorescence intensities of your standard and your new DNA prep should confirm the OD measurement.
24. Store the DNA prep at 4°, -20° or -80°.
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