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Preparation of Total DNA from Plant Tissue
Efendi

MATERIALS
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
 
METHODS
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
 
 
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Contact: efendi@ige.tohoku.ac.jp