Sean Lauber:RNA Extraction Using Trizol

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Procedure

  1. Ensure your cells are alive (if they're dead you won't get any RNA!)
  2. Remove media on cells and add 1 ml of Trizol (regardless of plate size). DO THIS ON THE BENCH AND NOT IN THE TISSUE CULTURE HOOD
  3. Scrape cells and pipette into a 1.5 ml eppendorf
  4. Pipette up and down to lyse cells
  5. Incubate at RT for 5 min. At this point you can store the sample at -80*C or continue
  6. In a fume hood, add 200 μL chloroform, shake to mix
  7. Incubate at RT for 3 min
  8. Spin at 13K RPM/4*C/15 min
  9. Pipette 350 μL of the top (aqueous) layer into a new tube. If the aqueous layer become cloudy, spin down for another minute before pipetting.
  10. Add 350 μL RNAse-free isopropanol, invert several times to mix
  11. Incubate RT for 10 min
  12. Spin 13K RPM/4*C/10 min
  13. Pour the isopropanol out (it's okay if there's some left over)
  14. Wash the RNA pellet: add 1 ml 75% RNAse-free EtOH, invert several times, spin at 9K RPM/4*C/5 min
  15. Carefully pipette off supernatant while avoiding the pellet; spin down again to remove the residual EtOH. Be aware of where the pellet is (depending on how you placed your tubes in the centrifuge) and pipette on the opposite side to avoid disturbing it. If the pellet falls off the wall of the tube, spin it down again and carefully remove the EtOH.
  16. Incubate tubes with the caps open at 55*C until all the EtOH has evaporated. Be careful not to let the pellet dry out (it will be harder to resuspend). Monitor your tubes and when there's no EtOH left, promptly remove them from the heat source.
  17. Resuspend the pellet in 10 μL of DEPC-treated water by pipetting up and down (do not vortex).
  18. Dissolve the pellet at 55*C for 10 min.
  19. Store at -80*C if required
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