... kit to the room temperature (20 - 25 °C ) for at least 30 min, note that each reagent must be ... with the cover membrane, incubate at 25 °C for 30 min .4. Pour liq ui d out of microwell, flap ... L/well of tri- deionized water , wash for 4-5 times , marinate for 10 s every time, then take out and flap ... manuall y , and incubate at 25 °C for 15 min in the dark for coloration .6. Determination: add 50 µ L ...