DNA Miniprep Isolation from Plants

Description

Procedure
1. Place 0.5 to 2 g (1 g is ideal) of plant tissue in a mortar and add an excess of liquid nitrogen. As the liquid nitrogen evaporates, grind the tissue thoroughly into a fine powder.

2. Transfer the plant powder into a 15 ml tube and add 7 ml of Extraction Buffer.

3. Mix the solution well using a spatula or a glass rod.

4. Cap the tubes and vigorously shake.

5. Incubate at 65°C for at least 5 min.

6. Add 2.5 ml of 5 M Potassium Acetate.

7. Cap the tubes and vigorously shake.

8. Incubate on ice for at least 15 min.

9. Centrifuge the tubes at 4°C for 10 min at 8,000 X g.

10. Add 5 ml of Isopropanol to a new 15 ml tube.

11. Prepare Miracloth™ filter funnel (see #1).

12. Using a 5 ml pipette, slowly filter the supernatant (prepared in Step #9) through the Miracloth™ filter.

13. Add the filtered supernatant to the 15 ml tubes containing Isopropanol, cap tubes and invert several times to precipitate the DNA (see #2).

14. Centrifuge tubes at room temperature for 10 min at 8,000 X g.

15. Remove the supernatant from the tube and add en equal volume of 80% Ethanol.

16. Mix vigorously by hand and centrifuge tube as in Step #14.

17. Remove the supernatant.

18. 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.

19. Redissolve pellet in 500 ìl of TE Buffer.

20. Transfer the DNA solution to a new microcentrifuge tube.

21. Precipitate the DNA with 50 ìl of 3 M Sodium Acetate and 500 ìl of Isopropanol.

22. Mix well and centrifuge in a microcentrifuge briefly (30 sec) at maximum speed.

23. Remove the supernatant and wash the DNA pellet by adding an equal volume of 80% Ethanol, mix well, centrifuge as previously and decant the supernatant.

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

25. Redissolve the DNA pellet in 200 to 300 ìl of TE Buffer.

26. Before using the DNA, centrifuge in a microcentrifuge at maximum speed for approximately 30 seconds to pellet any insoluble material.

Recipes
3 M Sodium Acetate

80% Ethanol 80% (v/v) Ethanol

5 M Potassium Acetate

TE Buffer 10 mM Tris, pH 8.0
1 mM EDTA

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*

Supplies

Tips
1. Cut Miracloth™ filter paper into approximately 4 cm2 pieces. With a 15 ml tube in a tube rack, push a Miracloth™ filter into the opening with a pipette tip. Do not push the filter paper too far down into the mouth of the tube. Slowly apply the supernatant onto the filter paper and allow for gravity filtration.

2. You can eliminate both centrifugation steps (Step #14 and #16) if you remove the DNA by using a glass hook. While the DNA is still on the hook, use a squirt bottle, and wash the DNA pellet with 80% Ethanol. After washing for a couple of seconds, place the DNA into a new microcentrifuge tube. Return to protocol Step #18.
(责任编辑：泉水)

搜索

热门标签:

DNA Miniprep Isolation from Plants