Yeast RNA Isolation
- Grow 50 ml of yeast to mid-log phase (OD600 0.8-2.0).
It is convenient to use a disposable 50-ml conical tube for steps 2-5.
- Spin down and resuspend yeast pellet in lysis buffer (10mM Tris-HCl pH 7.4, 10 mM EDTA, 0.5% SDS).
- Freeze by immersing the tube in liquid nitrogen, and store at -80°C until all samples are harvested.
- Thaw samples and add an equal volume of hot phenol (65°C, either water equilibrated or equilibrated with low pH buffer) and vortex vigorously.
- Incubate at 65°C for 30 minutes, vortexing every 5 minutes.
- Transfer to a 15-ml round bottom polypropylene tube.
- Spin in JA20 type rotor for 20 minutes at 10,000 rpm. Use appropriate bumpers.
- Remove top aqueous phase to a new tubes and add 1/10 volume of 3M sodium acetate, pH 5.3, and vortex.
- Add 2.5 volumes of 100% ethanol [or an equal (1.0) volume of isopropanol] and vortex.
- Store 1 hour to overnight at -20°C.
- Spin at 10,000 rpm for 20 minutes.
- Pour off supernatant carefully and wash pellet with 75% ethanol.
- Re-spin for 5 minutes. Carefully remove and discard supernatant.
- Let air dry, and resuspend in TE. Usually about 1 ml will give an RNA concentration of about 1-2 µg/µl.