YEAST HISTONE PREP (Modified, 2/17/2003)

Day 1: Inoculate a 25 ml culture in YPD (or selective media) at 9:00 and grow 9-10 hours, shaking, 30 C. Transfer to 1 liter YPD (or selective media), grow overnight Day 2: For analytical experiments, grow to OD600=1.0

1. Spin cells in large rotor (JA10), 5K g, 5 min.

2. Wash cell pellets in ~ 200-400 mls. sterile water and collect into one bottle. Re-spin.

3. Resuspend cells in 50 mls. DTT/Tris (pH 9.5). transfer to JA 14 tube, Incubate shaking, 30 C, 15 min.

4. Spin as above. Resuspend pellet in 50 mls. Sorbitol/Hepes (Buffer #2); Re-spin. Weigh the yeast cells.

5. Resuspend pellet in 50 mls. Buffer #2; add 2.75 mg zymolyase / g yeast cell. Incubate 30 C 45 min.

6. Add 100 mls. cold Buffer #3 (Sorbitol/Pipes/MgCI2). Spin 3.5K g in JA14, 4 C, 5 min.

Rest on ice

7. Resuspend pellet in 50 mls. ice cold NIB (1 mM PMSF), and hold in ice/water for 20 min. Spin 4K g, JA-14, 5 min, 4 C.

8. Repeat NIB wash 2X, each for 20 min on ice water.

9. Resuspend pellet in 50 mls. A wash(1 mM PMSF) and hold in ice water 15 min. Spin 4K g, 5 min.

10. Repeat A wash (15 min) and spin 4K g, 5 min.

11. Resuspend pellet in 50 mls. B wash(1 mM PMSF),. Hold on ice water for 5 min. Spin 4K g, 5 min.

12. Resuspend in 25 mls. B and transfer to JA 20 tube, Spin 4K g, 5 min. 4 C.

13. Resuspend pellet in 3volumes of cold H2O, add 5 N HCl to final concentration of 0.25 N to extract histones. Hold in ice water for 30 min, vortex occasionally.

14. Spin 30K g, 10 minutes. SAVE SUPERNATANT which contains the extracted histones. Measure volume with pipette.

15. Add 100% TCA to a final concentration of 20%. A large precipitate should form almost immediately. Hold on ice water for 30 min.

16. Spin 30 K g in JA 20 for 30 min (leave brake off!). If supe still looks cloudy, pour into a fresh tube and spin again. Wash pellet(s) in 10 mls. Cold acid acetone (0.5% HCl acetone) and spin 5 min, 30 K g. Wash pellet in cold acetone and spin again, 30 K g, 5 min. Pour off acetone carefully and air dry pellet(s).

17. Resuspend pellets in 250 ul of 10 mM Tris, pH 8.0. If hard to resuspend, add more Tris, 0.1 ml at a time and try again to resuspend. Try to keep histones as concentrated as possible.

18. Store histones at -20 C. Run 15 and 30 ul on a 15% SDS gel to check concentration and cleanliness of prep.

Buffers (Volume required for 1 sample)

1. DTT/Tris: 0.1mM Tris: HCI, pH 9.4 , 10 mM DTT (50ml)

2. 1.2 M Sorbitol , 20 mM HEPES , pH7.4 (100ml)

3. 1.2 M Sorbitol , 20 mM PIPES , 1 mM MgCl2, pH 6.8 (100ml)

4. NIB: 0.25 M sucrose , 60 mM KCl, 15 mM NaCl , 5 mM MgCl2, 1 mM CaCl2 , 15 mM MES, pH6.6 , 1 mM PMSF , 0.8% Triton X-100 (150ml)

5. 'A' wash: 10 mM Tris, pH 8.0 , 0.5% NP 40 , 75 mM NaCI , 30 mM NaButyrate , 1 mM PMSF (100 ml)

6. 'B' wash: 10 mM Tris, pH 8.0 , 0.4 M NaCI , 30 mM NaButyrate , 1 mM PMSF (75 ml)

7. 5N HCl

8. 100 % TCA