Posthybridization Washes

Steps 1-7 are performed at the hybridization temperature. Steps 8-11 are performed at room temperature.

1. Wash the embryos for 10 min in 50% formamide, 5X SSC.

2. Wash for 10 min with 0.5 mL of a 3:1 mixture of 50% formamide, 5X SS :2X SSC.

3. Wash for 10 min with 0.5 mL of a 1:1 mixture of 50% formamide, 5X SSC:2X SSC.

4. Wash for 10 min with 0.5 mL of a 1:3 mixture of 50% formamide, 5X SSC:2X SSC.

5. Wash for 10 min in 1 mL of 2X SSC, 0.01% Tween-20 alone at the hybridization temperature.

6. Wash for 30 min in 1 mL of 0.2X SSC, 0.01% Tween-20 at the hybridization temperature. This is best done on the shaker in the hybridization oven. Alternatively, use a heating block or water bath, but gently invert the tube every 5-10 min.

7. Repeat the stringent wash in step 6.

8. At room temperature, wash for 10 min in 1 mL of a 3:1 mixture of 0.2X SSC:PBT.

9. At room temperature, wash for 10 min in 1 mL of a 1:1 mixture of 0.2X SSC:PBT.

10. At room temperature, wash for 10 min in 1 mL of a 1:3 mixture of 0.2X SSC:PBT.

11. Replace the solution with PBT.

Was this article helpful?

0 0

Post a comment