Table 3.
Nick Translation Schema for Labeling Multiplex FISH Probes
| Fluorophore-labeled dUTP and dTTP concentrations | |||
|---|---|---|---|
| dUTP | EX/EM, nm | 0.2 mmol/L dUTP, μL∗ | 0.1 mmol/L dTTP, μL† |
| 0.2 mmol/L Aqua | 433/480 | 3 | 4.5 |
| 0.2 mmol/L Green | 497/524 | 3 | 4.5 |
| 0.2 mmol/L Gold | 550/564 | 3 | 4.5 |
| 0.2 mmol/L Orange | 559/588 | 2.5 | 5 |
| 0.2 mmol/L Red | 580/603 | 2.5 | 5 |
| 0.2 mmol/L Cy5 | 650/662 | 3 | 4.5 |
| MIX for nick translation reaction | |||
|---|---|---|---|
| Reagent | Volume, μL | ||
| 0.2 mmol/L dUTP | ∗ | ||
| 0.1 mmol/L dTTP | † | ||
| 0.1 mmol/L dNTP | 20 | ||
| 10× Nick translation buffer | 10 | ||
| Total | 45 | ||
| Nick translation reaction components (in duplicate) | |||
|---|---|---|---|
| Probe label | Amount (×2 tubes) | ||
| DNA | 1 μg | ||
| dH2O | 17.5 μL, volume of DNA | ||
| MIX | 22.5 μL | ||
| Nick translation enzyme | 10 μL | ||
| Total | 50 μL | ||
Table depicting the quantities of each of nick translation reaction components. Labeled dUTP is selected and added to the master mix. Nick translation is performed by combining 1 μg of DNA, the master mix, and the nick translation enzyme.
dH2O, distilled water; EM, emission; EX, excitation; FISH, fluorescence in situ hybridization; MIX, solution mixture.
∗
As specified in fluorophore-labeled dUTP concentrations.
†
As specified in fluorophore-labeled dTTP concentrations.