DNA Staining Protocol for Microarrays using POPO3

Material:

• P-3584 POPO -3 iodide (534/570) *1 mM solution in DMSO* (Molecular Probes)
• 1X Phosphate-buffered saline (PBS)
• 2x SSC
• 70% EtOH
• 90% EtOH
• 100% EtOH

1. Dilute the POPO3 1:5,000 in PBS up to a total volume of 100 µl*
2. Transfer this solution to a coverslip
3. Lower the microarray slide towards the coverslip until the droplet touches the slide surface
4. By lowering the microarray slide a bit more, the coverslip will stick to your slide
5. Turn around the microarray slide, so the coverslip is faced upwards
6. Place the microarray slide in the dark for 30 minutes
7. Wash the slide in 2x SSC until coverslip falls of the microarray slide
8. Wash the slide for 3 minutes in 2x SSC
9. Equilibrate your slide in 70% EtOH for 3 minutes
10. Equilibrate your slide in 90% EtOH for 3 minutes
11. Equilibrate your slide in 100% EtOH for 5 minutes
12. Air-dry your slide
13. Scan your slide using the Cy3 channel of your scanner

• When using large microarrays, you might have to adjust the volume.

We graciously thank Rolf Turk, Ellen Sterrenburg, Stefan White, Ralph Florijn and Johan den Dunnen of the Netherlands Leiden University Medical Centre for providing this protocol.