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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
 

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 l 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 4C. Resuspend pellet in 2 mls of ice cold HE buffer and transfer to 2 microfuge tubes. Spin
for 30 seconds at top speed at 4C. Keep pellets on ice for immediate use or store at -80C.
2. Resuspend cell pellet in 250 l 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 4C for 30 minutes.
4. Freeze in powdered dry ice for 10 minutes (or longer).
5. Microfuge for 5 minutes at top speed at 25C. 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 4C.
6. Transfer ~200 l 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 -20C. 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 4C. 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

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine