title: Loblolly pine 26K cDNA array v2.0, PtGen2
status: Public on Jan 26 2011
contact.city: Athens
contact.first: Adam
contact.middle: W.
contact.last: Barb
contact.email: abarb@uga.edu
contact.state: GA
contact.address: 180 E. Green Street
contact.department: Biochemistry and Molecular Biology
contact.country: USA
contact.institute: University of Georgia
organism: Pinus taeda
manufacture_protocol: Plasmid DNAs that had been propagated in the EST sequencing pipeline were re-arrayed and consolidated into new plates to using a BioMek 2000 liquid handling robot (Beckman Coulter, Fullerton, CA).  Samples were then diluted five-fold in millipure dH20 (mp dH20) and 5mL was transferred to a 96-well plate (ABgene AB-0900, Thermo Fisher Scientific Inc. Waltham, MA) for PCR amplification.  PCR reactions were performed in a 100mL volume containing 10 mM Tris (pH 8.8), 3.5 mM MgCl2, 25 mM KCl, 0.25 mM (ea.) dNTP, 0.4 mM pSLFOR2 primer {5’-AAAACGACGGCCAGTGCCAAG-3’}, 0.4mM pSLREV2 primer {5’-GCTTCCGGCTGCTATGTTGTGTGG-3’}, and 1 mL of homemade Taq polymerase.  PCR reactions were incubated at 95oC for 3 min., followed by 30 amplification cycles (95oC for 30 sec., 58oC for 1 min., 72oC for 2.5 min.) and then incubated at 72oC for 7 min.  Each reaction product was verified by agarose gel electrophoresis prior to samples being vacuum desiccated in a Savant Speed Vac Plus Model SC210A (Thermo Fisher Scientific Inc., Waltham, MA).  Samples were resuspended in 30mL spotting buffer (150mM PO₄ 1.5M Betaine, 0.001% SDS) with a Robbins Scientific HydraTM96 liquid handling robot and 15mL was transferred to 384 well Corning Costar spotting plates 07-200-747(Corning Inc., Corning, NY).  Printing was done at the Vanderbilt Microarray Shared Resource (VMSR) facility.  cDNAs were spotted onto Corning UltrGAPS slides (Corning Inc., Corning, NY) and printed with a BioRobotics Microgrid II (Genomic Solutions, Ann Arbor, MI) with a 48 pin print head