A set of 3,308 oligonucleotides were designed with Primer 3 (code available at http://www.genomewi.mit.edu/genome_software/other/primer3.htm) to amplify, via PCR, an internal fragment from each of the 1,654 predicted open reading frames (ORFs) identified in the annotated genomic sequence of C. jejuni NCTC 11168. Genomic DNA (20 ng) was used as a template in the first round of PCR amplifications with standard methods in a 96-well plate format. The PCR products were analyzed by agarose gel electrophoresis. Successful PCR products were reamplified to reduce the amount of residual genomic DNA carried over from the first PCR. PCRs without product or with incorrectly sized products were performed again by modifying the reaction conditions or by designing new primers. DNA fragments were obtained for approximately 98% of the ORFs. PCR products were purified with the Millipore PCR96 cleanup kit and quantified with the PicoGreen double-stranded DNA quantitation reagent from Molecular Probes. They were diluted in a 50% dimethyl sulfoxide solution at a concentration of 75 ng/µl and rearrayed into a 384-well format. They were then printed on aminosilane-coated glass microscope slides (CMT GAPS-II; Corning Inc., Corning, N.Y.) with an arrayer robot (Molecular Dynamic). Finally, the DNA fragments were immobilized onto the slides by baking at 80°C for 4 h.
Less...
More...