RNA Extraction From Yeast Using Glass Beads
· In advance, prepare 2.0 ml tubes containing 0.25 g acid washed glass beads (0.5 mm diameter)
and 250 µl ØOH:CHCl3:isoamyl alcohol (25:24:1). Label and place on ice.
1. For standard preparation of total RNA from yeast, collect 5 OD600 units of yeast (e.g., 10 mls of
OD600 = 0.5). Spin in IEC centrifuge for 5 minutes at 2000 rpm at 4°C. Resuspend pellet in 2
mls of ice cold DEPC-treated HE (10 mM HEPES, 1 mM EDTA, pH 8) and transfer to two microfuge
tubes. Spin for 30 seconds at top speed at 4°C. Store pellets on ice for immediate use, or at -80°C.
For pulse-chase labeled cells, start with 0.5 OD600 unit of yeast per time point. Collect cells by
centrifugation. Store cell pellets on ice for immediate use, or at -80°C until needed for extraction.
2. Resuspend cell pellet in 250 µl HENS buffer. Quickly transfer to 2.0 ml tube with glass beads and
ØOH:CHCl3:IAA. Vortex for 10 seconds. Do one tube at a time. Place tube(s) on ice.
HENS buffer: 10 mM HEPES-NaOH, pH 7.5 Treat with DEPC.
1 mM EDTA
300 mM NaCl
3. Place tubes in multi-tube vortex mixer at 4°C. Vortex for 10-30 minutes (25-75% breakage).
4. Microfuge for 10-30 minutes at 4°C at top speed. If the interface is broad, spin longer.
5. Transfer 200 µl of the supernatant to a 1.5 ml microfuge tube. Do not collect any of the interface.
· For pulse-chase experiments, collect exactly the same volume of supernatant from each tube.