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Yeast RNA Isolation

Method

  1. 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.

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

© 2014 The University of Texas MD Anderson Cancer Center