1. Chop a small amount of plant material (typically 20 mg) with a new razor blade or a sharp scalpel in 0.5 ml of ice-cold Otto I buffer in a petri dish.
2. Add 2 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.
3. Mix well with a pipette.
4. Filter the suspension through a 42 µm nylon mesh.
5. Store at room temperature, analyzing within 5 - 15 min.
6. Analyse relative DNA content of isolated nuclei.
Browning due to phenolic compounds may be inhibited by adding 2µl/ml ß-mercaptoethanol to Otto II buffer prior its use.
This procedure gives good results only with some species. If the results are not satisfactory, it is recommended to test a standard two-step procedure.
1. Chop a small amount of plant material (typically 20 mg) with a new razor blade or a sharp scalpel in 0.5 ml of ice-cold Otto I buffer in a petri dish.
2. Add 0.5 ml of ice-cold Otto I buffer, mix well with a pipette.
3. Filter the suspension through a 42 µm nylon mesh.
4. Incubate for 1 - 5 min, shake occasionally.
5. Add 2 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.
6. Store at room temperature, analyzing within 5 - 15 min.
7. Analyse relative DNA content of isolated nuclei.
If necessary, the samples can be kept at room temperature for prolonged periods of time after step 4 ( prior to the addition of Otto II buffer).
Browning due to phenolic compounds may be inhibited by adding 2µl/ml ß-mercaptoethanol to Otto II buffer prior its use.
This procedure gives good results only with some species. If the results are not satisfactory, it is recommended to test a standard two-step procedure.
