Oligonucleitide Hybridization to Glass Arrays
1. (optional) Prescan slide in Array Scanner II to determine quality of slide (Note: I usually only do this in the green channel (532nm) to save time.
2. Prepare 0.3 to 1 pmole of each Cy3 or Cy5 labeled primer in 50Âµl 1X hybridization solution (50% formamide, 5X SSC, 0.1% SDS, 100Âµg/ml salmon sperm DNA).
3. Pipet 50Âµl hyb solution with probe onto long edge of slide.
4. Cover with long cover slip, avoid air bubbles (gives high background).
5. Incubate overnight (12hrs) at room temperature in humid chamber (moist paper towel in bottom of relatively airtight container).
6. To remove coverslip, submerge slide in 2X SSC/0.2% SDS.
Turn on scanner now if you want to scan immediately after processing, scanner needs at least 40 minutes to warm up.
7. Wash 5 min in 2X SSC/0.2% SDS at room temperature.
8. Wash10 min at room temp with 2X SSC/0.1% SDS.
9. Rinse slide 10 seconds in ddH20.
10. Dry with compressed air.
11. Store in dark until ready to scan.
12. Scan in Array Scanner II in both channels (make sure scanner has had at least 40 minutes to warm up).