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DNAzol Prep

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