Isolation of RNA from Bacteroids

1. Grind 3 g nodules (fresh or frozen in liquid N₂) to a powder in mortar and pestle with liquid N₂.
2. To the powder, add ice-cold 0.5 M mannitol (or sucrose), 0.05 M Tris (pH 7.5), 0.02 M Na succinate, 5 mM Na dithionite.
3. Pass the resuspension through one layer of cheesecloth and rinse the cloth with 5 mL of the same buffer solution.
4. Spin filtrate in a sterile centrifuge tube and spin 5 min at 1500 rpm.
5. Respin supernatant for 5 min at 9000 rpm.
6. Resuspend pellet in LiCl buffer containing 10 mM vanadyl ribonucleosides and treat as stated above.

Reagents

• 0.1 M LiCl
• 1% SDS
• 7 M urea
• 0.1 M Tris (pH 8.4)
• 5 mM EDTA

Extract with phenol: chloroform 3 times. Precipitate with 2 vol ethanol. Spin. Redissolve precipitate in 2 mL H₂O treated with 0.1% DEPC and autoclaved. Add 6 mL 4 M Na acetate (6) treated with DEPC. Incubate on ice 2 hr, spin 30 min at 8000 rpm, and wash precipitate 3 times with ice cold 3 M Na acetate (6) treated with DEPC. Redissolve RNA in DEPC-treated H₂O, precipitate with 2.5 vol ethanol, and store as precipitate at –80°C.