Phenol/Freeze RNA Prep
Schmitt et al. (1990) NAR 18, 3091-3092.
Grow 10 ml of yeast to 5E6 cells/ml.
Harvest cells by centrifugation and resuspend cells in 400 ul of AE buffer.
Transfer cells to a 1.5 ml eppendorf tube and add 40 ul of 10% SDS. Vortex.
Add equal volume of phenol, vortex, and incubate at 65oC for 4 minutes.
Rapidly chill tube in dry ice/ethanol bath until phenol crystals appear, and centrifuge at room temperature for 2 minutes.
Transfer aqueous phase to a new tube, add an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1), vortex, and spin for 5 minutes at room temperature.
Transfer aqueous phase to a new tube, add 1/10 volume of 3M NaOAc, pH 5.3, 2.5 volumes of ethanol, and then precipitate for 20 minutes at -20oC.
Pellet RNA, wash with 80% ethanol, air dry pellet, and resuspend in 20 ul water.
Glass Bead RNA Prep
Grow cells to no later than late log phase (2E7 cells) in 20 ml of media (protocol can be scaled up or down)
Transfer cells to 50 ml conical tube. Spin 3000 rpm in Beckman table top for 5 minutes.
Wash pellet with DEPC-treated distilled H2O.
Spin 3000rpm 5 min. Can store pellet at -70oC indefinitely.
Resuspend pellet in 0.6 ml RNA extraction buffer.
IMMEDIATELY add an equal volume of phenol:chloroform:isoamyl alchohol (50:50:1). Mix.
Let sit at room temperature 5-6 minutes.
During this time, transfer to 13 x 100 mm glass tubes.
Add glass beads to ~3/4 up to organic phase meniscus. Vortex 2 minutes at max speed.
Transfer solution to 1.5ml eppendorf tube. Separate phases by centrifugation.
Extract aqueous phase twice with phenol:chloroform:isoamyl alchohol (50:50:1), then once with chloroform:isoamyl alcohol (24:1). (Add equal volumes each time).
Add 0.1 volume (approx. 40 ul) 3 M NaOAc pH 5.2 and 2-3 volumes cold 95% EtOH. Precipitate at -20oC for 1 hour.
Pellet, and wash pellet with 70% EtOH.
Resuspend with DEPC-treated distilled H2O (~30 ul). Store at -70oC.
Add DEPC to 0.1%, mix overnight, autoclave to destroy DEPC
50 mM NaOAc, pH 5.3, 10 mM EDTA)
TO MAKE 500 ml:
Final pH should be 5.3. Use DEPC-treated distilled H2O.
- 3.4 g NaOAc
- 10 ml 0.5 M EDTA stock solution
RNA Extraction buffer (0.1 M NaCl, 10 mM EDTA, 5% SDS, 50 mM Tris pH 7.5)
TO MAKE 100 ml:
Tris cannot be DEPC treated, so just use an autoclaved solution.
- 2.0 ml 5 M NaCl (DEPC-treated) stock solution
- 25 ml 20% SDS (DEPC-treated) stock solution
- 2.0 ml 0.5M EDTA (DEPC-treated) stock solution
- 5.0 ml 1M Tris pH 7.5 stock solution