Schizosaccharomyces pombe Total RNA Preparation

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Contributor:

J. Michael Bishop
University of California, San Francisco


Procedure:


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).



Solutions


5.7 M CsCl
192 g CsCl
200 ml total volume, pH 7.0
40 ml 0.5 M EDTA



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



30% (w/v) Sarkosyl

0.5 M EDTA

2 M Tris, pH 7.5

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



YPD
Autoclave for 20 min and cool to room temperature
10 g/liter Yeast Extract
20 g/liter Glucose
20 g/liter Peptone



TE buffer, pH 7.0
10 mM Tris
1 mM EDTA
pH 7.0



70% (v/v) Ethanol



BioReagents and Chemicals:


Sodium Chloride
Peptone
Yeast Extract
Sarkosyl
Cesium Chloride
Ethanol
2-Mercaptoethanol
Glucose
EDTA
Sodium Phosphate, Dibasic
Tris


Protocol Hints:


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