- Extract buffer:
- 100mM Tris HCI (pH8.0)
- 500mH NaCI (14.61)
- 50mH EDTA.2Na.2H2O (pH 8.0)
- Mercaptoethanol
- 20% SDS
- 5M K-acetate
- Potassium acetate
- 5.1 -TE buffer:
- 50mH Tris (pH8.0)
- 10mM EDTA (pH8.0)
- 80% ethanol
- Leaves (1 g)
- Liquid nitrogen
- Sterilized mortar and pestle
- Sterilized tubes (4) ml)
- Pipetman 20, 200, 1.000 ul
- Sterilized Pipetman holder 20, 200, 1000 ul
- 1. Crush leaves in mortar with liquid nitrogen
- 2. Homogenize by 15 ml extract buffer
- 3. Fill in sterilized tube and put label
- 4. Add 10 ul mercapto ethanol (15M)
- 5. Add 1 ml 20% SDS
- 6. Incubate at 65oC for 10 minutes
- 7. Add 5 ml 5M K-acetate then vortex
- 8. Incubate at 0°C for 20 min (on ice)
- 9. Centrifuge at 14500 rpm and 4°C for 20 min (2500xg)
- 10. Through miracloth (~9 x 9 cm)
- 11. Add 10 ml isopropanol stored in -20°C
- l 2. Incubate at -20°C for 20 minutes
- 13. Centrifuge at 13.000 rpm for 15 minutes (20000xg)
- 14. Pellet on paper towel for 10 minutes
- 15. Add 700 ul 5.1-TE buffer
- 16. Transport to eppendorf tube (1.5 ml)
- 17. Centrifuge at 14.000 rpm for 10 minutes
- 18. Add 75 ul 3M Na-acetate
- 19. Add 500 ul isopropanol and mix
- 20. Incubate at -80°C for 5 - 10 minutes
- 21. Centrifuge at 14.000 rpm for 5 minutes
- 22. Wash the pellet by 80% Ethanol and vortex
- 23. Centrifuge at 14.000 rpm for 5 minutes
- 24. Dry by room condition for 10 minutes
- 25. Dry in vacuum for 5 minutes
- 26. Resolve by 50 - 200 ul TE buffer
- 27. Digest 2 ul DNA with 2 ul restriction enzyme