Fabrication of the TOM1 Microarray

Modified by Rob Alba on 02/12/04

Twelve-thousand-eight-hundred-sixty EST clones representing ~8,500 independent tomato loci were inoculated into 384-well plates containing LB+Amp and incubated for 24-48 hrs at 37 ^oC. Resultant cultures were used to inoculate duplicate PCR reactions, which contained 10 mM Tris-Cl (pH 9.2), 25 mM KCl, 3.5 mM MgCl2, 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dCTP, 0.5 mM dUTP, 0.04 uL Taq polymerase, 200 nM T3 primer, and 200 nM T7 primer. PCR reactions were incubated at 95 ^oC for 2 min, followed by 39 amplification cycles (94 ^oC for 20 sec, then 52 ^oC for 20 sec, then 72 ^oC for 1.5 min), and then incubated at 72 ^oC for 10 min. Products from duplicate 15 uL PCR reactions were combined and purified using a Genesis RSP 200 Liquid Handler (TECAN Inc., NC) and 384-well filter plates (# S384PCR10; Millipore Inc., MA). Purified products were dehydrated under vacuum, re-suspended in 12 uL of spotting buffer (3X SSC, 1.5 M betaine), and printed onto glass slides coated with g-amino-propyl-silane (25.3 X 75.5 mm, UltraGAPS; Corning Inc., NY) using a MicroGrid Pro arrayer (BioRobotics Inc., MA) with 32 MicroSpot2500 printing pins. Temperature and humidity inside the arrayer were maintained at 18-21 ^oC and 35-45% RH, respectively. Dwell time, spots per visit, pin wash time, and pin dry time were set at 1 second, 27 spots, 7 seconds, and 10 seconds, respectively. cDNA was fixed to the slides by treatment with 300 mJoules of UV irradiation followed by a two-hour incubation at 85^oC. Array fabrication was completed with a 2 minute wash in 0.2% SDS, three rinses in Milli-Q^® water, and a final rinse in 90% EtOH. EtOH was removed immediately via centrifugation (2 min at 500 rpm) and resulting microarrays were stored in a dust-free plexiglass chamber (~21^oC, 0% RH).