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Star Republic: Guide for Biologists

Hybridization

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Protocol I

1. Resuspend Cy-labeled sample in 15 ml hybridization bufer (50% formamide, 3X SSC, 1% SDS, 5X Denhardt's, and 5% dextran sulfate).

2. Denature at 80 C for 10 minutes.

3. Apply to array slide, cover with a coverslip.

4. Hybridize at 42 C in a humidified chamber for overnight.

5. Wash 2 minutes in 2X SSC, 0.1% SDS.

6. Wash 2 minutes in 1X SSC.

7. Rinse in 0.2X SSC.

8. Dry by centrifugation at 500 rmp for 5 minutes. Or dry with an air blower, blowing parallel to the slide surface. Don't let the wash solution dry on the slide, which will result in high background.

9. Detect using an array scanner.

Protocol II

1a. Resuspend Cy-labeled sample in 15 ml hybridization bufer (3.4X SSC, 0.3% SDS).

2a. Denature at 100 C for 2 minutes. Centrifuge at maximum speed for 15-20 minutes.

3a. Apply to array slide, cover with a coverslip.

4a. Hybridize at 42 C in a humidified chamber for overnight.

5a. Wash 2 minutes in 2X SSC, 0.03% SDS.

6a. Wash 2 minutes in 2X SSC.

7a. Wash 2 minutes in 1X SSC.

8a. Rinse in 0.2X SSC.

9a. Dry by centrifugation at 600 rmp for 2 minutes. Or dry with an air blower, blowing parallel to the slide surface. Don't let the wash solution dry on the slide, which will result in high background.

10a. Detect using an array scanner.