Friday, November 21, 2003

Description
The original maize DNA miniprep protocol is used extensively for many plant species and different tissues. This slightly modified version is acceptable for most DNA extractions. The procedure has the advantage of isolating DNA from plant material very rapidly. The procedure requires a table-top drill-press (mechanized homogenizer).

Procedure
1. Connect a microcentrifuge pestle to a homogenizer.

2. Place a small amount of leaf tissue in the microcentrifuge tube.

3. Add Liquid Nitrogen to the tube and allow the Liquid Nitrogen to evaporate.

4. Quickly grind the leaf tissue to a fine powder.

5. Add Extraction Buffer to the tissue powder and bring the volume to approximately 750 l (see #1).

6. Vortex the solution to suspend the plant material in the solution.

7. Incubate at 65C for 10 min.

8. Add 200 l of 5 M Potassium Acetate.

9. Cap the tubes and vigorously shake.

10. Incubate on ice for 20 min.

11. Centrifuge the tubes in a microcentrifuge for 10 min at room temperature and maximum speed.

12. Transfer the supernatant to a new microcentrifuge tube.

13. To the supernatant add an equal volume of Isopropanol and mix well.

14. Immediately centrifuge in a microcentrifuge at maximum speed for 2 min.

15. Discard the supernatant and wash the DNA pellet by adding an equal volume of 80% Ethanol, mixing well and centrifuge in a microcentrifuge at maximum speed for 2 min.

16. Discard the supernatant.

17. Invert the tubes on a paper towel for a couple of seconds to allow excess Ethanol to run out of the tube. Then air dry for a couple of minutes.

18. Redissolve pellet in 50 l of TE Buffer.

19. Recommendation: Pass the DNA through CL-6B microcentrifuge column to remove insoluble material before using DNA in polymerase chain reactions.

Recipes
3 M Sodium Acetate

80% Ethanol 80% (v/v) Ethanol


5 M Potassium Acetate

Extraction Buffer 1% (w/v) SDS
100 mM NaCl
10 mM EDTA
50 mM Tris, pH 8.0
*Add just before use
10 mM 2-Mercaptoethanol*


TE Buffer 10 mM Tris, pH 8.0
1 mM EDTA



Supplies


Tips
1. Wash the pestle clean of plant debris as you are adding the extraction buffer