Yeast Glass Bead RNA Prep

1. Set up tubes. Need 3 tubes for each prep.
        • tube 1: contains 400 µL of phenol/chloroform.
        • tube 2: contains 400 µL of chloroform.
        • tube 3: contains 40 µL of 3 M NaOAc (made with DEPC'd H2O)
2. Resuspend frozen cell pellet in 150 µL LET.
3. Add 150 µL phenol/LET.
4. Add 1 scoop full of glass beads.
5. Vortex in MultiMixer for 5 minutes on top speed.
6. Add 250 µL DECP'd H2O and 250 µL phenol/chloroform/LET.
7. Vortex well. Spin 2' in room temperature microfuge.
8. Remove top layer. Add to tube #1. Vortex well. Spin 2' in room temperature microfuge.
9. Remove top layer. Add to tube #2. Vortex well. Spin 2' in room temperature microfuge.
10. Remove top layer. Add to tube #3. Add 1 ml 100% ethonol. Freeze at -80ºC.
11. Spin 10' in room temperature microfuge. Wash pellet with ice cold 70% ethanol.
12. Spin 5' in room temperature microfuge. Drain supernatant. Dry pellet in speed vac (usually takes 10 to 15 minutes).
13. Resuspend pellet in 100 µL DEPC'd H2O.
14. Count 1:200 dilution in spec. to determine concentration.
    

LET Recipe

25 mM Tris, pH 8.0 100 mM LiCl         (all reagents should be made in DEPC'd H2O!) 20 mM EDTA