FGRS: protocol

 

Total RNA Preparation

Last Updated

January 10, 2012 10:17 AM


Materials


  1. 3M Sodium Acetate (pH 5.2)

  2. 20% SDS, stock solution

  3. Acid Phenol
    Stock solution, in 4°C refrigerator

  4. Chloroform
    Stock solution, in 4°C refrigerator

  5. Phase Lock Tubes, Heavy 2 mL (yellow)

  6. 70%, 100% EtOH

  7. AE Buffer
    50 mM Sodium Acetate (pH 5.2)
    10 mM EDTA (pH 8.0)


From the Iyer Lab


  1. 1.Spin down yeast cells (10-50 ml at OD 600 ≤ 1)  4000 rpm for 2 minutes.

  2. 2.Remove as much media from the cell pellet as possible.

  3. 3.Re-suspend cells in 400 µl AE buffer.

  4. 4.Transfer re-suspended cells to a 1.5 mL tube with a screw cap and rubber gasket.

  5. 5.Add 35 µl 20% SDS.

  6. 6.Add 400 µl acid phenol (pH 4.5-5.5)
    Be very sure to use acid phenol for this step.
    Be sure the cap is very tight.

  7. 7.Vortex cells at full speed for 30 s.

  8. 8.Incubate 1 h at 65°C, vortexing every 10 min.

  9. 9.Incubate on ice for 10 minutes.

  10. 10.Spin down at 10,000 rpm for 5 minutes.

  11. 11.Transfer aqueous (top) phase to a new 1.5 ml tube (avoiding the interphase) and add 400 µl of acid phenol.

  12. 12.Mix well by inverting tube.

  13. 13.Spin down at 10,000 rpm for 5 minutes.

  14. 14.Prepare a 2 ml heavy (yellow) phase lock tube by spinning at 12,000 rpm for 5 min.

  15. 15.Transfer aqueous (top) phase (avoiding the interphase) to the heavy phase lock tube.

  16. 16.Add 400 µl of chloroform and mix well by inverting the tube (do not vortex).

  17. 17.Spin down at 12,000 rpm for 5 minutes.

  18. 18.Dump supernatant into a new 1.5 ml tube.

  19. 19.Add 45 µl 3M sodium acetate (pH 5.2).

  20. 20.Add 900 µL 95% (or 100%) ice cold ethanol.

  21. 21.Precipitate for 15 min-overnight at -20°C.

  22. 22.Spin RNA pellet down full speed for 15 min at 4°C.

  23. 23.Remove ethanol completely with a L200 pipette tip.

  24. 24.Wash pellet with 500 µl of 70% ethanol.

  25. 25.Spin RNA pellet down full speed for 5 min at 4°C.

  26. 26.Pipette off all possible ethanol (this may take several re-spins and pipettes).

  27. 27.Allow to dry, re-suspend in ~50 µl RNase-free water.
    This is the only time the RNA is not kept on ice.

  28. 28.Measure concentration with NanoDrop.

  29. 29.Run 2 to 4 µg on a 1% agarose gel in order to check for degradation.