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The Laboratory of J. Michael Bishop at the University of California, San Francisco
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1. Grow an overnight culture of S. pombe in YPD (50 ml).
2. Inoculate 1 liter of prewarmed YPD with 50 ml of the overnight culture of S. pombe.
3. Grow at 37°C until the Optical Density at 600 nm (OD600) is 0.75 to 1.0.
4. Prepare and autoclave acid-washed glass beads (see protocol for the Preparation of Acid-Washed Glass Beads).
5. Pellet the yeast cells for 15 min in a JA10 rotor at 10,000 rpm (11,000 X g).
6. Resuspend the yeast in 50 ml of cold PBS.
7. Centrifuge the yeast for 15 min in a JS4.2 rotor at 3,000 rpm (1,900 X g).
8. Add an equal volume of acid-washed glass beads to the cells.
9. Vortex and shake vigorously for 5 min to break open cells.
10. Add 20 ml hot (65°C) GTC Solution and vortex well.
11. Pellet beads and cell debris in a JS4.2 rotor for 10 min at 3,000 rpm (1,900 X g).
12. Overlay the supernatant onto 17 ml of 5.7 M CsCl in 0.1 M EDTA.
13. Centrifuge at 25,000 rpm in a SW28 rotor (83,000 X g) at 20°C for at least 15 hr.
14. Remove most of the liquid from the tube and pour off the last 5 ml.
15. Cut the tube open 1 to 2 cm from the bottom.
16. The RNA should be visible as an opaque pellet.
17. Wash the RNA pellet with 0.5 ml cold 70% ethanol.
18. Resuspend pellet in 0.5 ml TE, pH 7.0 and add directly to 1 ml of Ethanol.
19. Precipitate and wash pellet with 70% Ethanol.
20. Resuspend the RNA in 1 ml TE, pH 7.0 and measure the absorbance at 260 nm.
21. Store the RNA as an Ethanol precipitate (See Hint #2).
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| TE buffer, pH 7.0 |
| 10 mM Tris
1 mM EDTA
pH 7.0
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| 70% (v/v) Ethanol |
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| 5.7 M CsCl |
| 192 g CsCl
200 ml total volume, pH 7.0
40 ml 0.5 M EDTA
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| 6 M GTC Solution |
| bring the volume to 200 ml with ddH2O
7.5 ml 2 M Tris, pH 7.5
3 ml 2-Mercaptoethanol
20.0 ml 30% Sarkosyl
142 g Guanidinium Thiocyanate (CAUTION! see Hint #1)
6.0 ml 0.5 M EDTA
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| 30% (w/v) Sarkosyl |
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| 0.5 M EDTA |
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| 2 M Tris, pH 7.5 |
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| PBS |
| pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl
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| YPD |
| Autoclave for 20 min and cool to room temperature
10 g/liter Yeast Extract
20 g/liter Glucose
20 g/liter Peptone
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Glucose
EDTA
Tris
Sodium Phosphate, Dibasic
Sodium Chloride
Peptone
Yeast Extract
Sarkosyl
Cesium Chloride
Ethanol
2-Mercaptoethanol
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. A 1 liter S. pombe culture at an OD600 of 1 yields approximately 1 mg of total RNA
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