# 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