Summary: Total RNA From Yeast Using Glass Beads
In advance, prepare 2 ml tubes containing 0.25 g acid washed glass beads (0.5 mm diameter) and
250 µl phenol:CHCl3:isoamyl alcohol (25:24:1). Label and place on ice.
1. Collect 5 OD600 units of yeast (e.g., 10 mls of OD600 = 0.5). Centrifuge for 5 minutes at 2000 rpm
at 4°C. Resuspend pellet in 2 mls of ice cold HE buffer and transfer to 2 microfuge tubes. Spin
for 30 seconds at top speed at 4°C. Keep pellets on ice for immediate use or store at -80°C.
2. Resuspend cell pellet in 250 µl HENS buffer. Quickly transfer to 2 ml tube with glass beads.
Close tube quickly but carefully (glass beads can interfere with cap closure). Vortex for 10
seconds. Place tube on ice. Do one tube at a time.
3. Parafilm tubes to secure tops. Vortex in multi-tube mixer at 4°C for 30 minutes.
4. Freeze in powdered dry ice for 10 minutes (or longer).
5. Microfuge for 5 minutes at top speed at 25°C. The tube will thaw and an interface layer will
separate the upper aqueous and lower phenol layers. If the interface is broad, spin longer at 4°C.
6. Transfer ~200 µl of aqueous supernatant to a 1.5 ml microfuge tube. Do not collect any interface.
7. Repeat extraction of aqeuous layer. Add equal volume of phenol:CHCl3:IAA. Vortex 15 seconds.
Spin 5 minutes. Transfer supernatant to fresh 1.5 ml tube.
8. Add 3 volumes of 100% EtOH to supernatant. Precipitate 1 hour to overnight at -20°C. Do not
add additional salt (e.g., don't add NaOAc, pH 5). Optional: DEPC treat EtOH.
9. Spin for 30 minutes at top speed at 4°C. Carefully remove supernatant. Pellet may be small.
10. Wash pellet with of 75% EtOH (prepared with DEPC treated dH2O). Spin for 5 minutes at top