Microarray printing was performed at the Machines for Genes Laboratory, CSIRO Livestock Industries (Geelong, Vic). Of the 20 384-well source plates, 19 were printed in duplicate side-by-side, whilst the 384-well control plate was printed in duplicate at both the top and bottom of each printed block, giving a total of 16,128 printed elements. Elements were spotted on Corning UltraGAPS slides using a BioRobotics MicroGrid II TAS using MicroSpot 2500 pins. These pins produce a spot size of approximately 100 μm diameter with a pitch of 220 μm between spots. Each amplified DNA fragment was printed in duplicate adjacent to each other. The pin configuration was arranged in 4 columns and 12 rows yielding 48 subarrays of 19 × 19 spots. Elements were printed in a 150 mM sodium phosphate printing buffer (pH 8.5). The spots were then fixed to the slide by baking for two hours at 80°C. To ensure DNA spotting was uniform across the slides, samples were taken from each batch of microarrays and probed with Panomer™ 9 random oligodeoxynucleotide conjugated with Alexa Fluor® 532 (Molecular Probes, Invitrogen). Reproducibility was visually assessed between spot duplicates and between slides within a printing batch.
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