- Grind tissue, in liquid nitrogen, to a fine powder. (100-250mg per 300ul DNAzol)
- Scrape tissue with pre-cooled metal spatula into tube containing DNAzol
- Mix by gentle inversion a few times and incubate at room temp with shaking for five minutes. (When doing multiple preps, invert samples every few minutes)
- Add 300ul Chloroform (per 300ul DNAzol in step one), shake vigorously for 30 seconds, then shake for five minutes.
- Centrifuge at 12,000 Xg for 10 minutes (* Can stop after this step, and store up 1 month at 4 C)
- Transfer supernatant to a new tube.
- Add 3ul Rnase A (per 300ul DNAzol), mix, and incubate for 20 min at 37 C.
- Add to aqueous phase 225ul 100% EtOH (per 300ul DNAzol) and mix by inverting 6-8 times, incubate 5 minutes at room temp.
- Spin at 5,000 Xg for 4 minutes, discard supernatant. (Should see pellet, but not always)
- Wash pellet with 300ul Plant DNAzol/ EtOH mix, vortex. (a. 1 vol Plant DNAzol : 0.75 vol 100% EtOH or b. 171.4 ul DNAzol : 128.6ul EtOH)
- Incubate at room temp for 5 minutes.
- Spin at 5,000 Xg for 5 minutes, pour off supernatant
- Wash with 1ml 75% EtOH, vortex
- Spin at 5,000 Xg for 2 minutes, pour off supernatant. (then quick spin, and pipet remaining EtOH)
- Air dry pellet for 5-10 minutes
- Re-suspend DNA pellet in 50ul TE (LTE or ddH2O can be used as well)