Glass Bead RNA Prep (Yeast)

Tuesday, November 18, 2003

Description
Glass Bead RNA Prep (Yeast)

Procedure
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. 40l) 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 l). Store at -70 deg C.

Recipes
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

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Glass Bead RNA Prep (Yeast)