The commercial Zebrafish oligo library from Compugen/Sigma-Genosys consists of 16,399 oligos (65nt) representing 16,288 unique gene clusters. The oligos are modified with a 5' amino C6 linker. The library includes 172 zebrafish beta-actin oligos distributed over the entire library as internal controls (approximately 4 per 384-well plate). Amersham Code Link activiated glass silides were prepared using a hydrophillic polymer containing N-hydroxysuccinimide ester groups covalentely attached to the silane base coat by the vendor. The oligos were resuspended in 50mM phosphate buffer, pH 8.0, at 20mM concentration. The individual oligo probes were printed on Code Link activated slides under 45% humidity by Gene Machine Omni Grid 100 Microarrayer in 4 X 12 pin configuration and 20 x 20 spot configuration of each subarray. The spot diameter was 100um and distance from center to center was 200um. The printed Zebrafish microarrays were further immobilized via convalent coupling methods at 70% humidity overnight. Samples were hybridzed after additional 100mM Taurine/Bicine blocking and 4X SSC/0.1% SDS washing steps.
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