RNA preps

Fatima Cvrckova FATIMA at AIMP.UNA.AC.AT
Wed Aug 3 02:16:53 EST 1994


Dear Doug et al.,

I never tried any fancy $68 kit for isolating RNA from yeast, but everybody
in our lab is now happily using the simple, fast and reliable protocol
published by Cross and Tinkelenberg, Cell 65:875-883, 1991. The method
follows:

EXAMPLE - making RNA from cycling cells:

Start with cca 20 ml of YEPD cultures O.D. cca 0.7 - 0.8. (they can be obtained 
by diluting an overnight inoculum 0.25 ml into 20 and 4-5 h cultivation - as 
for spheroplasts).

Put crushed ice (cca 15 ml incl. free space) into 50 ml Falcon tubes and pour 
the cultures onto it. Spin at 4 degrees 1500 rpm 2 min (Sigma centrifuge), 
discard supernatant.

Resuspend pellets into cca 1 ml of ice-cold TE, transfer into 2.2 ml safelock 
Eppendorf tubes. Spin for 10s in the coldroom and discard supernatant.

To the pellet add: 
1. cca 200 µl of glass beads
2. 350 µl of 50:49:1 mixture of phenol: chloroform: isoamylalkohol (isoamylalk. 
could be omitted?) equilibrated w. TE
3. 350 µl of Cross RNA buffer 1

Close the tubes and seal the lids with parafilm (to avoid having the phenol 
everywhere in the next step). Put the tubes into glass tubes and shake 
vigorously on the Ika-Vibrax-VXR mixer in the coldroom for 10 min.

Spin for 5 min in an angle microfuge and transfer the upper phase (carefully, 
w/o interphase!) into 1ml of precooled -20 degrees ethanol in 1.5 ml Eppendorf 
tubes. Mix and leave at -20 for 10 min.

Spin in the angle microfuge in the coldroom for 5 min full speed. Dissolve 
sediment in Cross RNA buffer 2 (acc. to the amount of RNA - e.g. 100 µl) by 10 
min incubation at 65 degrees. Measure RNA concentration. Store in -20.

I got cca 250 - 300 µg from 20 ml of 1107 cells.

Cross RNA buffer1:
0.3 M NaCl
10 mM Tris pH 7.5
1 mM EDTA
0.2 % SDS
Sterility NOT essential since this is to be added to phenol. Keep in 4 degrees 
and don't worry when SDS precipitates out; shake the buffer well before use and 
try to get as homogenous suspension as possible.

Cross RNA buffer 2:
1x TE + 0.2 % SDS
Sterility ESSENTIAL. DEPC-treated water recommendable. Keep at room temp.

NOTE: This method works well also with frozen cells.

Good luck!

Fatima



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