DNA extraction from "recalcitrant" plant tissues

  1. Put 70 mg of tissue in a mortar, add liquid nitrogen and grind gently to fine powder with a pestle. Warm up to room temperature before going to step 2.
  2. Add 2 ml of Buffer 1 and homogenize. If the slurry gets very sticky start over using less tissue. Transfer homogenate to 2 ml Eppendorf tube.
  3. Spin down for 5 min at room temperature (15000 g).
  4. Discard supernatant and suspend pellet in 1.7 ml of Buffer-1.
  5. Spin down suspension for 5 min at room temperature (15000 g). If the supernatant is still very sticky repeat steps 4-5.
  6. Discard supernatant and suspend pellet in 0.4 ml of Buffer-2.
  7. Add 20 µl of of 20% (w/v) N-lauroylsarcosine, keep for 15 min at room temperature.
  8. Add 0.4 ml of CTAB solution, incubate at 62°C for 30 min.
  9. Add 10 µl of 10 mg/ml Proteinase K, incubate at 37°C for 30 min.
  10. Add equal volume of CIA (chloroform-isoamyl alcohol, 24:1) and invert gently for 10 min.
  11. Spin down for 10 min at room temperature (15000 g), collect upper layer to a new tube.
  12. Add 3 µl RNAse (10 mg/ml), shake briefly, incubate for 30 min at 37°C.
  13. Repeat steps 10-11.
  14. Add 2/3 volume of ice cold isopropanol and incubate for 15 min at 4°C to precipitate DNA.
  15. Spin down for 10 min at room temperature (15000 g).
  16. Discard supernatant and wash twice DNA pellet with 70% ethanol.
  17. Air dry and suspend in 50-100 l TE Buffer, shake briefly, and leave on shaker for 30 min at room temperature or overnight at 4°C.
  18.   Buffer – 1

    Buffer – 2

    Lauroylsarcosine Solution

    Make to 100 ml with dH2O.

    CTAB Solution

    Make to 100 ml with dH2O.

    TE Buffer

    Adjust pH 8.0 with HCl


    by Alfred E. Szmidt