title: Cornell-CGEP Tomato 13K vTOM1
status: Public on Nov 09 2005
contact.city: Bet-Dagan
contact.first: Carmiya
contact.last: Bar-Or
contact.email: carmiya@agri.gov.il
contact.address: Hamaccabim
contact.department: Genomics
contact.country: Israel
contact.institute: ARO
contact.phone: 972-3-9683034
organism: Solanum lycopersicum
manufacture_protocol: 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 85oC. 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 (~21oC, 0% RH).