Isolation of RNA from Free-Living Rhizobia

Cells from 100 mL culture in early-log-phase GYPC were spun and resuspended in 1 mL sterile 25% sucrose. This solution was transferred to a sterile centrifuge tube and 6.5 mL M-STET, 0.5 mL lysozyme (10 mg/mL), and 0.4 mL 200 mM vanadyl ribonucleoides were added.

M-STET: 4% sucrose, 6% triton X100, 0.06 M Tris (pH 8), 0.06 M EDTA Incubate on ice for 5 min. Boil 2 min (or add SDS/sarkosyl, extract with phenol:chloroform, and precipitate), chill, and spin 10 min at 8000 rpm. To the supernatant, add 0.7 vol isopropanol, chill, spin again, and resuspend pellet in 5 mL.

5X Running buffer for RNA (formaldehyde) gels: (for 500 mL) 2 mL 0.5 M EDTA, 6.8 g Na acetate, 12.8 g MOPS (free acid), 9.0 g MOPS (base).

1. Induce cells normally –10 mL of 0.2 OD600.
   Wash cells (grown overnight in ORS minimal plus yeast at 30° or 37°C) 2 times with nif media and diluting to 0.2 OD (10 mL each). Check 1 bottle for induction by acetylene reduction.
2. After induction, add 0.5 mL vanadyl ribonucleosides directly through the septum into a vial with a syringe (vanadyl protects cells against oxygen).
3. Put cells in plastic tubes and spin for 2 min at 8000 rpm in Sorvall.
4. Resuspend in 10 M urea lysis buffer with a plastic pasteur pipette.
5. Add 0.5–0.75 mL of phenol:chloroform (50:50) (“phenol” is phenol: m-cresol:hydroxyquinoline) to the microfuge tube.
6. Vortex, place at 65°C for 2–3 min. Repeat. Vortex and spin 2–3 min in microfuge.
7. Re-extract the liquid phase 2 more times at room temperature with phenol:chloroform. Avoid proteinaceous interphase when removing the upper liquid phase.
8. Extract with chloroform 1 time.
9. Precipitate with 100% ethanol, 0.3 M Na acetate (5.2) at –20°C overnight.
10. Collect precipitate, wash 1 time with 70% ethanol, 1 time with 100% ethanol, and resuspend in 10 mM Na acetate (5.2). Treat for gels.