Phenol/Freeze RNA Prep

 

Phenol/Freeze RNA Prep

Schmitt et al. (1990) NAR 18, 3091-3092.

DEPC is used to rid solutions of RNases. DEPC is very toxic and should be used only in the hood.

Stuff you need:
(DEPC treatment means: add DEPC to 0.1%, mix O/N, autoclave to destroy DEPC)

DEPC treated water

AE buffer (DEPC treat)
50mM Na acetate, pH 5.3
10mM EDTA

10% SDS

Phenol, equilibrated with AE buffer

Phenol:chloroform:isoamyl alcohol, 25:24:1

3M Na acetate pH 5.3 (DEPC treat)

RNase-free 95% EtOH (-20 deg C)

RNase-free 80% EtOH

1. Grow 10 ml of yeast to 5E6 cells/ml.

2. Harvest cells by centrifugation and resuspend cells in 400 µl of AE buffer.

3. Transfer cells to a 1.5 ml microcentrifuge tube and add 40 µl of 10% SDS. Vortex.

4. Add equal volume of phenol, vortex, and incubate at 65 deg C for 4 minutes.

5. Rapidly chill tube in dry ice/ethanol bath until phenol crystals appear, and centrifuge at room temperature for 2 minutes.

6. Transfer aqueous phase to a new tube, add an equal volume of phenol/chloroform/IAA, vortex, and spin for 5 minutes at room temperature.

7. Transfer aqueous phase to a new tube, add 1/10 volume of 3M Na Acetate, pH 5.3, 2.5 volumes of ethanol, and then precipitate for 20 minutes at -20 deg C.

8. Pellet RNA, wash with 80% ethanol, air dry pellet, and resuspend in 20 µl water.

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Glass Bead RNA Prep

DEPC is used to rid solutions of RNases. DEPC is very toxic and should be used only in the hood.

Stuff you need:
(DEPC treatment means: add DEPC to 0.1%, mix O/N, autoclave to destroy DEPC)

DEPC treated water

RNA extraction buffer
0.1 M NaCl (DEPC treat)
10mM EDTA (DEPC treat)
5% SDS (DEPC treat)
50mM Tris-HCl, pH 7.5 (CANNOT DEPC treat)

PCI
50:50:1 of phenol:chloroform:isoamyl alcohol

SEVAG
24:1 chloroform:isoamyl alcohol

3M NaOAc pH 5.2 (DEPC treat)

RNase-free 95% EtOH (-20 deg C)

RNase-free 70% EtOH

1. Grow cells to no later than late log phase (1E7 cells) in 20 ml of media (protocol can be scaled up or down).

2. Transfer cells to 50 ml Falcon tube. Spin 3000rpm in Beckman table top for 5 min.

3. Wash pellet with DEPC treated water.

4. Spin 3000rpm 5 min. Can store pellet at -70 deg C indefinitely.

5. Resuspend pellet in 0.6 ml RNA extraction buffer.

6. IMMEDIATELY add an equal volume of PCI . Mix.

7. Let sit at RT 5-6 min.

8. During this time, transfer to 13x100 mm glass tubes.

9. Add glass beads to ~3/4 up to organic phase meniscus. Vortex 2 min at max speed.

10. Transfer solution to 1.5ml microfuge tube. Separate phases by centrifugation.

11. Extract aqueous phase 2X with PCI, then 1X with SEVAG. (Add equal volumes each time).

12. Add 0.1 volume (approx. 40µl) 3M NaOAc pH 5.2 and 2-3 vols cold 95% EtOH. Ppt. at -20 deg C for 1 hr.

13. Pellet, and wash pellet with 70% EtOH.

14. Resuspend with DEPC treated water (~30 µl). Store at -70 deg C.