On the basis of these sequence analysis and annotation results, we developed a cDNA microarray consisting of 4,793 non-redundant clones. Prior to printing, we prepared a cDNA insert of each clone. Briefly, E. coli containing the clones was grown overnight in GS-96 medium (BIO 101 Systems, Carlsbad, CA) followed by incubation of 10 μl of cell culture plus 90 μl of water at 95°C for 10 min to release plasmid DNA. Ten microliters of supernatant liquid containing plasmid DNA was added to a PCR cocktail containing Taq DNA polymerase in a total volume of 100 μl. The mixtures were denatured at 95°C for 3 min and then subjected to 35 cycles of denaturation (95°C, 30s), annealing (55°C, 30s), and extension (72°C, 2 min). The products were ethanol-precipitated, washed, and reconstituted in 20 μl of TE buffer. Thirty microliters of 80% dimethyl sulfoxide was added, and the mixture was printed on CMT-GAPS-coated slides (Corning, NY) using an OmniGrid MicroArrayer OGR-03 (Genomic Solutions, San Carlos, CA). Each cDNA clone was printed in duplicate on each slide. After arraying, slides were UV-linked, washed
