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Arabidopsis Oligonucleotide Microarrays |
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Troubleshooting the microarrays
The 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.Specific Problems:
1. I do not see any hybridization signals from the microarrays.
- Make sure the slides do not contact the hot water surface during rehydration.
- Make sure your labeling reaction is optimized (we generally see color in the final elution of target).
- Remember to boil the labeled target before adding it to the microarray.
- Remember the arrays comprise sense strands of DNA. Conventional RNA amplification techniques may produce sense targets which should not hybridize.
2. I see hybridization signals but they are very faint.
- Increase the number of times (4-5) that you perform the rehydration step over the hot water bath.
- Double the UV energy employed for crosslinking (from 120 to 240 mJ).
3. I see hybridization signals at the edges but not in the center of the microarrays.
- Make sure you are using a hybridization volume of 200-250 microliters, and place two small strips (1-1.5 mm wide x 40-50 mm long) cut from the plastic cover slips, at the edges of the microarrays, to provide a raised coverslip configuration. Here is an illustration of this.
4. I see a big glow with excessive background.
- Make sure you thoroughly purify the labeled target (we generally purify it four times).
Page last updated February 18, 2003