Posthybridization Washing

Embryos are sequentially washed in solutions of increasing stringency to remove nonspecifically bound probe.

1. Transfer the embryos to a 1.5-mL microcentrifuge tube using a Pasteur pipet.

2. Discard as much of the old hybridization solution as possible, then briefly rinse and then wash the embryos in fresh hybridization solution (4X SSC, 0.1% Tween-20, 50% formamide) for 20 min at room temperature. Use approx 0.5-1 mL of wash solution for all washes and gently agitate the embryos (e.g., on a rotating wheel).

2. Wash in 4X SSC, 0.1% Tween-20, 40% formamide for 20 min at room temperature.

3. Wash in 4X SSC, 0.1% Tween-20, 30% formamide for 20 min at room temperature.

4. Wash in 2X SSC, 0.1% Tween-20, 20% formamide for 20 min at room temperature.

5. Wash in 2X SSC, 0.1% Tween-20, 10% formamide for 20 min at room temperature.

0 0

Post a comment