Yeast RNA Extraction Using Hot Phenol
· See Köhrer and Domdey, 1991, Methods Enzymol., 194:398-405. Note: Prepare dry ice
powder in bucket. Preheat phenol/AE to 65°C. Heated phenol should not be re-used.
1. Collect 5-10 OD600 units of yeast (e.g., 1 ml of OD600 = 5-10). [Up to 25 OD600 units may be
used with this method.] For rRNA pulse-chase labeling time course, collect 1 ml of cells in a 2 ml
screw-cap microfuge tube containing 0.5 ml of 25% glycerol pre-cooled to -20°C.
2. Spin in microfuge 30 seconds at room temperature (RT). Discard supernatant (SN). Cell pellets
may be frozen at -70°C at this point.
3. Resuspend (R/S) pellet (PT) in 400 µl of AE buffer.
4. Add 40 µl 10% SDS. Vortex briefly. Quickly proceed to step 5.
5. Immediately add 500 µl hot phenol/AE (see below). Vortex for 15 seconds. Incubate at 65°C for 5
minutes. Vortex every 30 seconds for 5 seconds.
6. Cool to room temperature by placing tube in powdered dry ice for ~30 seconds. Do not cool below
7. Centrifuge 10 minutes in microfuge at 6000 rpm at room temperature.
8. Remove ~450 µl of lower phenol layer. Leave behind cell PT, interphase layer, and aqueous SN.
9. Repeat steps 5 - 7.
10. Transfer aqueous SN to fresh 1.5 ml microfuge tube. Estimate volume.
11. Extract with equal volume of phenol:CHCl3/ANE (see below). Vortex for 1 minute at RT. Spin for