Procedure: (all steps at 0°C–4°C unless indicated)
- 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).
- After washing (ice-cold H2O), chop into small pieces with a single-edge
- 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.
- 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.
- Filter through 4 layers of cheesecloth, then through 2 layers of Miracloth
- Centrifuge 9000 rpm for 15 minutes 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 2 30-mL Corex tubes at 8000 rpm for 10 minutes in a Beckman
- Repeat step 7, and centrifuge at 6000 rpm for 10 minutes in a JS-13 rotor.
- 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.
- 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
- Resuspend nuclei in 5 to 10 mL of 250 mM sucrose, 10 mM Tris-HCl
(pH 8.0), and 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 50–100 µg/mL and digest at 55°C until the solution clarifies (2 hrs).
- 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.
- 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