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- Harvest 100 mg tissue (about 1 leaf) and flash freeze in liquid nitrogen
- Add 1ml TRIzol
- Homogenize and incubate at room temperature for 5 minutes
- Add 200μl chloroform and shake for 15 seconds
- Incubate 2 minutes at room temperature
- Centrifuge 12000 rpm for 15 minutes at 4°C
- Transfer aqueous phase to new tube containing 500μl isopropanol
- Incubate 10 minutes at room temperature
- Centrifuge 12000 rpm for 10 minutes at 4°C
- Discard supernatant and wash pellet in 1 ml 75% ethanol (made with DEPC water)
- Centrifuge at 7000 rpm for 5 minutes at 4°C
- Air dry for 10 minutes
- Resuspend in 30μl DEPC water
- Incubate 10 minutes at 55°C
- Store at -80°C
- After step 13 add 3μl NaOAc
- Add 60μl ethanol 100%
- Incubate at -80°C for 30 minutes
- 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 tha next day
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