Nuclei isolation modified from Hamilton, Kunsch, and Temperli (1972) Anal. Biochem. 49:48-57; further modified by Tom Guilfoyle, then N. Olszewski and Eric Richards.

Procedure: (all steps at 0-4C unless indicated) Harvest 100 g tissue (plants can be any age up to just bolting), which has been destarched by placing in the dark for 48 hr). After washing (ice cold H2 O), chop into small pieces with a single-edge razor blade. Add ice cold diethylether until it covers the tissue and stir for 3 min., pour into Buchner funnel to remove ether, rinse with cold H2O. Add 300 ml of buffer A (1 M sucrose, 10 mM Tris-HCl (pH 7.2), 5 mM MgCl2 , 5 mM 2-mercaptoethanol and 400ug/ml ethidium bromide). Grind tissue with a Polytron (Brinkmann) at medium speed for 1-3 min. until tissue is homogenized. Filter through 4 layers of cheesecloth, then through 2 layers of Miracloth (Calbiochem). Centrifuge 9000 rpm for 15 min. in a Beckman JA-10 rotor. Resuspend pellet in 50 ml of buffer A plus 0.5% Triton X-100 (Sigma) with a homogenizer (55 ml glass pestle unit). Centrifuge in two 30 ml Corex tubes at 8000 rpm for 10 min. in a Beckman JS-13 rotor. Repeat step 7, centrifuge at 6000 rpm for 10 min in a JS-13 rotor. Resuspend pellet in 10 ml of buffer A plus 0.5% Triton X-100. Layer crude nuclei over two 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 , 34ul 2-mercaptoethanol, and Percoll to a final volume of 100 ml. Centrifuge in a JS-13 rotor at 2000 rpm after 5 min. increase speed to 8000 rpm and centrifuge an additional 15 min. Starch will pellet, the bulk of the nuclei will band at 35%-65% interface and intact cloroplasts will band at the 0-35% interface. The zone containing the nuclei is collected, diluted with 5-10 volumes of buffer A and pelleted by centrifugation at 8000 rpm for 10 min. in a JS-13 rotor. Nuclei can be visualized by light microscopy after staining with 1/5-1/10 volume 1% Azure C (Sigma) in buffer A minus ethidium bromide. Resuspend nuclei in 5-10 ml of 250 mM sucrose, 10 mM Tris-HCl (pH 8.0), 5 mM MgCl2 by homogenization. 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 50-100ug/ml and digest at 55C until the solution clarifies (2 hr). Add 21 g CsCl, when dissolved add 1 ml of 10 mg/ml ethidium bromide and transfer to two quick-seal Ti 70.1 tubes and centrifuge at 45000 rpm at 20C in a Beckman Ti70.1 rotor for 36-48 hours. Remove banded DNA, extract with 1 volume of 3 M CsCl saturated isopropanol, repeat extraction until all ethidium bromide is removed, and dialyze four times against 1 L of TE plus 10 mM NaCl.

Note: The inclusion of ethidium bromide is essential if high molecular weight DNA is desired (I. Rubensteins lab, Plant Phys. 66:1140 (1980).