Protocol 113 Hybridization and posthybridization procedure

1. Prepare PN buffer for the posthybridization washes to be performed tomorrow.

PN buffer (1000 ml):

Take 473.5 ml 0.2 M Na2HPO4/0.1% NP-40. Add 400 ml distilled water.

Adjust to 1000 ml with distilled water. Store at room temperature.

2. Prepare the hybridization station as suggested by the manufacturer.

3. Place the pretreated slide in the hybridization station.

4. Start the hybridization program:

Step 1. O-ring conditioning: 3 min 75°C

Step 2. Introduce probe: 37°C hold step until probe is loaded

Step 3. Hybridization: Overnight 37°C hold step (usually 18 h)

Step 4. Formamide wash: Temperature increases to 45°C followed by five cycles of 10-s flow time, 60-s hold time

Step 5. Preparation for PN buffer wash: Reducing temperature to 20°C

Step 6. PN buffer wash: Five cycles at 20°C of 30-s flow time, 60-s hold time

Step 7. PN buffer hold: One cycle at 20°C of 10-s flow time, 2-h hold time

NOTE: During the hold time of step 7, you can abort the program when you are ready to remove the slide from the station.

5. Remove the slide from the hybridization station and place it in a coplin jar filled with PN buffer.

6. Incubate the slide for 10 min.

7. Dip the slide in water and place it in a 50-ml falcon tube. Dry the slide immediately by centrifugation (216 g, 5 min, room temperature).

8. Slide is now ready for scanning. Keep the slide in the dark until use.

DNA microarrays: analysis of chromosomes and their aberrations

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