| Contributor: |
The Laboratory of Linda Breeden at the Fred Hutchinson Cancer Research Center
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1. Start with a 10 to 20 ml yeast culture at mid log-phase.
2. Pellet cells by centrifugation at 1500 X g for 10 min and discard the supernatant.
3. Resuspend the cells in 1 ml of cold RNA Buffer then repellet the cells by centrifugation at 1500 X g for 10 min and discard the supernatant.
4. Freeze pellet in dry ice and store it at -80°C if desired.
5. Thaw the cells on ice.
6. Add 100 to 200 μl ice-cold RNA Buffer and resuspend the pellet by vortexing.
7. Add 0.5 volume of Acid-Washed Glass Beads (see protocol for the Preparation of Acid-Washed Glass Beads).
8. Mix the cell/glass bead suspension by vortexing briskly at 4°C for 3 min.
9. Add 450 μl of room temperature RNA Buffer with SDS and mix by vortexing briefly.
10. Add 450 μl Water-Saturated Phenol.
11. Mix by vortexing briskly at 4°C for 3 min.
12. Centrifuge in a microcentrifuge at full speed for 10 min at 4°C.
13. Transfer upper aqueous phase to a fresh tube avoiding any of the interface.
14. Add 0.5 volume Water-Saturated Phenol and mix by vortexing.
15. Add 0.5 volume Chloroform (CAUTION! see Hint #1) and mix by vortexing.
16. Centrifuge for 2 min at full speed in a microcentrifuge. Recover the aqueous phase and repeat the Chloroform extraction and recover the aqueous phase.
17. Add 20 μl of 4 M NaCl and 1 ml of Ethanol to the aqueous phase.
18. Precipitate the RNA by incubation at -20°C to -80°C for 30 min.
19. Centrifuge in a microcentrifuge at full speed for 10 min at 4°C.
20. Remove supernatant and resuspend the pellet in 150 μl 70% Ethanol.
21. Centrifuge for 10 min at full speed in a microcentrifuge and remove the Ethanol.
22. Allow the pellet to air dry.
23. Resuspend the pellet in 30 to 50 μl ddH2O and store the RNA at -20°C to -80°C.
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| RNA Buffer |
| 100 mM NaCl
50 mM Tris, pH 7.4
10 mM EDTA
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| Water-Saturated Phenol (CAUTION! see Hint #1) |
| See protocol for the Preparation of Water-Saturated Phenol
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| 4.0 M NaCl |
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| RNA Buffer with SDS |
| 100 mM NaCl
50 mM Tris, pH 7.4
10 mM EDTA
1.3% (w/v) SDS
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Chloroform
Ethanol
Phenol
Tris
EDTA
Sodium Chloride
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
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