Arabidopsis Thaliana DNA Isolation

1. Harvest 100 g tissue (plants can be any age up to just bolting), which has been destarched by placing it in the dark for 48 hrs).
2. After washing (ice-cold H₂O), chop into small pieces with a single-edge razor blade.
3. Add ice-cold diethylether until it covers the tissue and stir for 3 minutes. Pour into the Buchner funnel to remove ether and rinse with cold H₂O.
4. Add 300 mL of buffer A (1 M sucrose, 10 mM Tris-HCl (pH 7.2), 5 mM MgCl2, 5 mM 2-mercaptoethanol, and 400 µg/mL ethidium bromide). Grind tissue with a Polytron (Brinkmann) at medium speed for 1 to 3 minutes until tissue is homogenized.
5. Filter through 4 layers of cheesecloth, then through 2 layers of Miracloth (Calbiochem).
6. Centrifuge 9000 rpm for 15 minutes in a Beckman JA-10 rotor.
7. Resuspend pellet in 50 mL of buffer A plus 0.5% Triton X-100 (Sigma) with a homogenizer (55-mL glass pestle unit).
8. Centrifuge in 2 30-mL Corex tubes at 8000 rpm for 10 minutes in a Beckman JS-13 rotor.
9. Repeat step 7, and centrifuge at 6000 rpm for 10 minutes in a JS-13 rotor.
10. Resuspend pellet in 10 mL of buffer A plus 0.5% Triton X-100. Layer crude nuclei over 2 discontinuous Percoll gradients constructed in 30-mL Corex tubes as follows: 5-mL layers containing from the bottom upward 60% (v/v) and 35% (v/v) percoll A:buffer A. Percoll A is made as follows: to 34.23 g sucrose add 1.0 mL of 1 M Tris-HCl (pH 7.2), 0.5 mL of 1 M MgCl₂, 34 µL 2-mercaptoethanol, and Percoll to a final volume of 100 mL centrifuge in a JS-13 rotor at 2000 rpm. After 5 minutes increase speed to 8000 rpm and centrifuge an additional 15 min. The starch will pellet, the bulk of the nuclei will band at a 35% to 65% interface and intact chloroplasts will band at the 0%–35% interface.
11. The zone containing the nuclei is collected, diluted with 5 to 10 volumes of buffer A, and pelleted by centrifugation at 8000 rpm for 10 minutes in a JS-13 rotor. Nuclei can be visualized by light microscopy after staining with 1/5 to 1/10 volume 1% Azure C (Sigma) in buffer A minus ethidium bromide.
12. Resuspend nuclei in 5 to 10 mL of 250 mM sucrose, 10 mM Tris-HCl (pH 8.0), and 5 mM MgCl₂ by homogenization.
13. Add EDTA to 20 mM and TE (10 mM Tris HCl (pH 8.0), 1 mM EDTA) to a final volume of 20 mL. Add 1 mL of 20% (w/v) Sarkosyl. Add proteinase K to 50–100 µg/mL and digest at 55°C until the solution clarifies (2 hrs).
14. Add 21 g CsCl. When dissolved, add 1 mL of 10 mg/mL ethidium bromide and transfer to 2 quick-seal Ti 70.1 tubes and centrifuge at 45000 rpm at 20°C in a Beckman Ti 70.1 rotor for 36 to 48 hours.
15. Remove banded DNA, extract with 1 volume of 3 M CsCl saturated isopropanol, repeat extraction until all ethidium bromide is removed, and dialyze 4 times against 1 L of TE plus 10 mM NaCl. The inclusion of ethidium bromide is essential if high molecular weight DNA is desired.