Table 2.
Four nuclear isolation buffers most frequently used in plant DNA flow cytometry
| Buffer | Composition* | Reference |
|---|---|---|
| Galbraith | 45 mm MgCl2, 30 mm sodium citrate, 20 mm MOPS, 0·1 % (v/v) Triton X-100, pH 7·0 | Galbraith et al. (1983) |
| LB01 | 15 mM Tris, 2 mm Na2EDTA, 0·5 mm spermine.4HCl, 80 mm KCl, 20 mm NaCl, 0·1 % (v/v) Triton X-100, pH 8·0† | Doležel et al. (1989) |
| Otto‡ | Otto I: 100 mm citric acid, 0·5 % (v/v) Tween 20 (pH 2–3) Otto II: 400 mm Na2PO4·12H2O (pH 8–9) | Otto (1992), Doležel and Göhde (1995) |
| Tris.MgCl2 | 200 mm Tris, 4 mm MgCl2.6H2O, 0·5 % (v/v) Triton X-100, pH 7·5 | Pfosser et al. (1995) |
*
Final concentrations are given.
†
The buffer formula contains 15 mm mercaptoethanol. However, as the other buffers were used without additives that suppress the negative effect of phenols and other cytosolic compounds, LB01 was used without mercaptoethanol.
‡
pH of the buffers is not adjusted. The nuclei are isolated in Otto I buffer; DNA staining is done in a mixture of Otto I and Otto II (1 : 2) with a final volume of 1 mL.