RNA Prep (TRIzol)

Material

  1. TRIzol Reagent [Invitrogen, Cat#. 15596026]

Method

  1. Pellet cells by centrifugation.
  2. Lyse cells in 0.5mL of TRIzol Reagent by repetitive pipetting in a 1.5mL tube.
  3. Incubate the homogenized samples for 5 minutes at RT.
  4. Add 0.1mL of chloroform and cap the tube securely.
  5. Shake tube vigorously by hand for 15 seconds.
  6. Incubate for 2-3 minutes at RT.
  7. Centrifuge the sample at 12,000 x g for 15 minutes at 4C. RNA remains exclusively in the upper aqueous phase.
  8. Transfer the aqueous phase to a fresh 1.5mL tube.
  9. Precipitate the RNA by using 0.25mL of isopropanol, 1/2 volume of TRIzol.
  10. Incubate samples at RT for 10 minutes.
  11. Centrifuge at 12,000 x g for 10 minutes at 4C.
  12. Remove the supernatant from the tube, leaving only the RNA pellet.
  13. Wash the RNA pellet once with 75% ethanol, adding at least 0.5mL 75% ethanol per 0.5mL of TRIzol.
  14. Vortex the sample briefly, then centrifuge the tube at 7,500 x g for 5 minutes at 4C and discard the wash.
  15. Air dry the RNA pellet for 5-10 minutes.
  16. Dissolve RNA in RNase-free water.