yeast RNA

FATIMA at AIMP.UNA.AC.AT FATIMA at AIMP.UNA.AC.AT
Wed May 5 05:46:00 EST 1993


(Stephanie Kong - kong1 at husc.harvard.edu - asking for yeast RNA)

Dear Stephanie and other interested people,
I am not sure how easy is sending yeast or any other RNA so that it stays
in a good shape. However, I can offer (and recommend!) a fast, reliable
and extremely simple protocol how to make good quality total yeast RNA
within half an hour. The resulting RNA gives decent, sharp and obviously
undegraded bands on Norterns - I never tried to do anything else with it.
The following method was published by Fred Cross and Arthur Tinkelenberg
(Cross and Tinkelenberg, Cell 65:875-883, 1991) and slightly modified in 
our lab.
 
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 microliters of glass beads
2. 350 microliters of 50:49:1 mixture of phenol: chloroform: isoamylalkohol 
(isoamylalk. could be omitted?) equilibrated w. TE
3. 350 microliters 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 
microliters) by 10 min incubation at 65 degrees. Measure RNA concentration.
Store in -20.

I got cca 250 - 300 micrograms from 20 ml of moreless wt (K1107) 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 Cvrckova (fatima at aimp.una.ac.at)



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