Maloof-\/\/ i K |

RNA extraction with TRIzol (invitrogen cat# 15596-026)

  1. Harvest 100 mg tissue (about 1 leaf) and flash freeze in liquid nitrogen
  2. Add 1ml TRIzol
  3. Homogenize and incubate at room temperature for 5 minutes
  4. Add 200μl chloroform and shake for 15 seconds
  5. Incubate 2 minutes at room temperature
  6. Centrifuge 12000 rpm for 15 minutes at 4°C
  7. Transfer aqueous phase to new tube containing 500μl isopropanol
  8. Incubate 10 minutes at room temperature
  9. Centrifuge 12000 rpm for 10 minutes at 4°C
  10. Discard supernatant and wash pellet in 1 ml 75% ethanol (made with DEPC water)
  11. Centrifuge at 7000 rpm for 5 minutes at 4°C
  12. Air dry for 10 minutes
  13. Resuspend in 30μl DEPC water
  14. Incubate 10 minutes at 55°C
    Store at -80°C

Extra steps to have a cleaner sample

  1. After step 13 add 3μl NaOAc
  2. Add 60μl ethanol 100%
  3. Incubate at -80°C for 30 minutes
  4. Centrifuge at maximum speed for 30 minutes at 4°C

1L DEPC water
Add 1ml DEPC to 1L milli-Q water
Let sit overnight
Autoclave the next day