DNA extraction from small amounts of plant tissue

  1. Warm 2x CTAB buffer to 60 C.
  2. Put 70 mg tissue in a mortar, add liquid nitrogen and grind gently to a fine powder. Warm up to room temperature.
  3. Add 1.5 ml of warm 2x CTAB buffer and homogenize gently. If the slurry gets very sticky start over with less tissue.
  4. Transfer homogenate to 2 ml Eppendorf tube, incubate at 62 C for 30 min.
  5. Add 10 l of 10 mg/ml Proteinase K, incubate at 37 C for 30 min.
  6. Add equal volume of CIA (chloroform-isoamyl alcohol, 24:1), vortex briefly and invert for 10 min.
  7. Spin down for 10 min (15.000 rpm). Collect top layer to new tube.
  8. Add 2.5 l RNAse (10 mg/ml), shake briefly, incubate at 37 C for 30 min.
  9. Add equal volume of CIA. Vortex briefly and invert for 5 min.
  10. Spin down for 10 min (15.000 rpm). Collect top layer to new tube.
  11. Add 2/3 volume of ice-cold isopropanol. Invert several times, incubate for 15 min at 4 C.
  12. Spin down for 10 min (15.000 rpm), with tube hinges facing away from center. Carefully remove supernatant.
  13. Wash DNA pellet twice with 250 m l of 70 % ethanol.
  14. Air dry DNA pellet.
  15. Add 50-100 m l TE buffer, shake briefly, and leave at shaker for 30 min or overnight at 4 C to resuspend.
  16. Run 3 m l DNA together with 1-2 m l 6x loading buffer on 0.8% agarose gel, to estimate DNA concentration.

2X CTAB Buffer

Make to 100 ml with dH2O.

TE Buffer

Adjust pH 8.0 with HCl

RNAse Stock solution (store at 20C)

Proteinase K stock solution (store at 20C)

Gel preparation and electrophoresis Run at 66 V for about 1 hour.

by Alfred E. Szmidt