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.
: 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
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).
- 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.
- After induction, add 0.5 mL vanadyl ribonucleosides directly through the
septum into a vial with a syringe (vanadyl protects cells against oxygen).
- Put cells in plastic tubes and spin for 2 min at 8000 rpm in Sorvall.
- Resuspend in 10 M urea lysis buffer with a plastic pasteur pipette.
- Add 0.5–0.75 mL of phenol:chloroform (50:50) (“phenol” is phenol:
m-cresol:hydroxyquinoline) to the microfuge tube.
- Vortex, place at 65°C for 2–3 min. Repeat. Vortex and spin 2–3 min in
- Re-extract the liquid phase 2 more times at room temperature with
phenol:chloroform. Avoid proteinaceous interphase when removing the
upper liquid phase.
- Extract with chloroform 1 time.
- Precipitate with 100% ethanol, 0.3 M Na acetate (5.2) at –20°C overnight.
- 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.