Construction of DNA Microarrays
The DNA microarrays have PCR fragments derived from genomic DNA corresponding to each protein-coding gene. High quality primers of 20-23 bases in length were picked for each gene by Steve Jones at the Sanger center in the U.K. The PCR fragments were one to two kb in length containing at least 700 bp of predicted protein coding sequence, or they contained more than 90% of the predicted coding sequence of the gene. None of the PCR fragments have known C. elegans repetitive elements, although there could be cross-hybridization between genes with highly similar sequences.
The PCR conditions were performed in 96-well plates as follows: 0.5uM each primer, 1x Perkin Elmer PCR buffer, 2mM MgCl2, 0.5% formamide, 50 ng/ul genomic DNA, 0.2mM dNTPs, 0.3ul Perkin Elmer Taq, in a 50ul reaction. The reaction conditions were 94 °C for 2min, followed by 30 cycles of 94 °C for 30 sec, 54-56 °C for 45 sec, 72 C for 1 min., followed by 5 min. at 72 °C. Aliquots of all PCR products were run on gels, and the PCR result for each gene was recorded if it showed one band of the correct size. The overall success rate of the PCR was about 88%. The PCR products were precipitated by adding either ethanol or isopropanol, and centrifugation for 30 minutes at 4 C at 4000rpm in a Sorvall RC-3B centrifuge. The ethanol/isopropanol was removed by aspiration or hand pipetting, and then the PCR products were washed with 70% ethanol and spun as before. The PCR products were allowed to dry overnight, suspended in 25 ul of 3 x SSC and re-arrayed into 384-well plates. Four 96-well plates were arrayed into five 384-well plates with each 384-well plate containing approximately 5ul of each PCR product.