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Summary: Auble Lab
Total Yeast RNA Prep
· Grow ~ 100 ml cells to OD600 ~1.0. Heat shock or treat if desired. Streak cells
on plates to confirm relevant phenotypes at the time of harvest.
· Pellet cells 6K X 5 min in GSA rotor; quick freeze in liquid nitrogen (or dry
ice/ethanol) and store pellets at 80 C or go to next step.
· Resuspend cells in 4 ml lysis buffer (10mM Tris-Cl pH 7.5, 10 mM EDTA, 0.5 %
SDS)
· Transfer cell suspension to sterile 15 ml conical tube; add 4 ml acid phenol (water
saturated, pH 4.3; Fisher BP1751I-400), parafilm around cap and vortex well.
· Incubate at 65 C for one hour with occasional vortexing.
· Place tubes on ice for 10 min, spin in clinical centrifuge at 4 C for 10 min.
· Remove aqueous layer, transfer to new sterile 15 ml conical tube, re-extract with
phenol (room temp, no incubation).
· Extract aqueous phase once with chloroform. Transfer aqueous phase to
chloroform washed, autoclaved 15 ml COREX tubes.
· Add 0.4 ml 3 M sodium acetate and 8 ml 200 proof ethanol. Incubate at 20 C for
~30 mins. Spin 8K X 20 min in SS34 rotor (with adaptors) at 4 C.
· Wash RNA pellets 3X with 8 ml 70% ethanol (store ethanol at 20 C).
· Dry RNA pellets briefly in speed-vac (need to remove speed vac rotor, parafilm
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