auxin preparation

This method describes the isolation of auxin from snapdragon tissue material as published by Okubo H et al. 1991.
  1. Collect fresh plant material (from 2 to 20 g).
  2. Lyophilize material
  3. Ground
  4. extract with 80% cold methanol at 5 degrees Celsius for 24h by shaking in the dark
  5. centrifuge
  6. filtrate
  7. reduce in vacuo at 37 degrees Celsius to aqueous solution
  8. extract four times with ethylacetate at pH 3.0
  9. extract ethylacetate phase four times with 2% sodium bicarbonate
  10. reacidify with HCl to pH 3.0
  11. extract four times with ethylacetate at pH 3.0
  12. evaporate the ethylacetate fraction to dryness
  13. adjust to 5% methanol with distilled water
  14. pass through a Sep-Pek C18 cartrigde (Waters Associates)
  15. wash cartridge with 90% methanol
  16. evaporate to dryness
  17. store frozen at -80 degrees Celsius