1a. Chop a small amount of plant material (typically 20 mg) with a new razor blade or a sharp scalpel in 1ml of ice-cold Otto I buffer in a petri dish.
1b. As an alternative, protoplasts can be prepared, and resuspended in ice-cold Otto I buffer to a concentration of 105 - 106/ml.
2. Filter the suspension through a 42 µm nylon mesh.
3. Pellet the nuclei (150g/5 min).
4. Remove the supernatant, leaving about 100 µl of the liquid above the pellet.
5. Resuspend the nuclei by gentle shaking. Add 100 µl of fresh Otto I buffer.
6. Incubate for 10 - 60 min, depending on species, at room temperature, shaking occasionally. Select the incubation period that gives the lowest background and CV.
7. Add 1 ml of Otto II buffer. It is preferable to include DAPI (or propidium iodide + RNase) in the Otto II buffer. Alternatively, these compounds can be added to the sample after the addition of Otto II buffer. The stains are used at the following concentrations: DAPI, 4 µg/ml; propidium iodide, 50 µg/ml + RNase, 50 µg/ml.
8. Store at room temperature, analyzing within 5 - 15 min.
9. Analyse relative DNA content of isolated nuclei.
Large numbers of samples can be prepared and simultaneously centrifuged (step 3). If necessary, the samples can be kept at room temperature for prolonged periods of time after step 5 (the addition of fresh Otto I buffer) prior to the addition of Otto II buffer and analysis.
Browning due to phenolic compounds may be inhibited by adding 2 µl/ml ß-mercaptoethanol to Otto II buffer prior its use.
In some species, a simplified procedure may be used.
