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1-Transfer 1.5 ml of liquid culture of yeast grown for 20 - 24 h at 30°C in YPD (1% yeast extract, 2% peptone, 2% dextrose) into a microcentrifuge tube. Pellet cells by centrifugation at 20,000 × g for 1-5 minutes
2- Ressuspend cells with 200 µl of TES, Add 400 µl of acid phenol and Incubate 1 hour 65C, vortexing every 10 minutes.
3- Incubate on Ice 10 min
4- Spin 5 min at 4 deg and transfer top layer to a new tube.
5- Add 400 µl acid phenol
6- Repeat step 4
7- add 400 µl chloroform, vortex and repeat step 4
8- Add 40 µl 3M Na Acetate, pH 5.3, 2.5 volumes of ethanol, repeat step 4, discart supernadant.
9- Wash pellet with 500 µl cold 80% ETOH, and air dry
10- Resuspend in 50 microliters H2O. Store RNA at –70 deg
TES:
0,5% SDS
10 mM Tris pH 7.5
10 mM EDTA
RNase-free water
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