Troubleshooting the microarraysThe rehydration and crosslinking step is critical and seems to cause the most difficulties. Rehydration should be done over (not in) a hot (50-60oC) water bath. The slide should be held DNA side down for ten seconds over the vapor coming from the surface of the water. Immediately dry the slide by placing it DNA side up on a heating block preheated to 65oC. Ensure the DNA elements do not touch anything during this process, which should be repeated 4-5 times. Rehydration is crucial since it ensures the maximum amount of DNA contacts the glass surface. Crosslinking is then done using a UV source. We generally use 120mJ, but this can be increased by a factor of two. Following crosslinking, wash the slides in 1% SDS and 100% ETOH, and then dry them, and use them for hybridization.
1. I do not see any hybridization signals from the microarrays.
2. I see hybridization signals but they are very faint.
3. I see hybridization signals at the edges but not in the center of the microarrays.
4. I see a big glow with excessive background.
Page last updated February 18, 2003