From Tsukiyama Lab, 2001
This is the preferred method for yeast RNA preparation
use Gloves and RNAse free solutions throughout.
1. Use a YPD overnight culture to innoculate fresh YPD media and grow cells at 30 degrees overnight.
Example: 120 microliters O/N in 800 ml YPD gives A600 ~0.85 after ~16 hrs.
2. For RNA samples from exponentially growing cells, take samples between A600 0.5 1.0. Remove the equivalent of 200 ml of A600 ~1.0 cells and centrifuge 4 min @ 4 deg. Resuspend in 10 ml ice cold water and transfer to 15 ml conical tube. Centrifuge 4 min @ 4 deg. Remove supernatant. Quickly resuspend pellet in 800 microliters cold TES and freeze in dry ice or liquid N2.
3. Warm acid phenol in H2O bath to 65 deg.
4. Thaw cells briefly on ice.
5. Add 800 microliters warm acid phenol and vortex 15 sec.
6. Incubate 65 deg for 60 min with vortexing every 10 min for 10 sec.
7. Divide evenly into two 1.5 ml microfuge tubes or one 15 ml tube.
8. Place samples on ice 5 min.
The following volumes are based on dividing the sample:
9. Spin 5 min at 4 deg and transfer top layer to a new tube.
10. Repeat acid phenol extraction until the interface is clean (~4 timesO.
11. Extract once with ~400 microliters CHCl3. Transfer top layer to a new eppendorf tube.
12. Ethanol precipitate by adding 40 microliters 3M NaOAc and 1 ml ETOH. Wash pellet in cold 80% ETOH twice.
13. Briefly dry pellet in speedvac. Resuspend in 50 microliters H2O. Store RNA at 70 deg.
Typical yield is ~300 micrograms RNA
Heat new bottle to 65 deg (100g).
Add 20 ml H2O + 0.2 g Hydroxyquinoline
Add ~10 ml more H2O till a little water remains on top of phenol so that it is completely water saturated.
10 mM Tris pH7.5
10 mM EDTA