title: UGA - BMB - Adams Lab, P. furiosus, 2192 ORFs x 3 Copies, Version 1
status: Public on May 25 2007
contact.city: Athens
contact.first: Michael
contact.middle: W. W.
contact.last: Adams
contact.state: GA
contact.address: 120 East Green St.
contact.department: Molecular Biology and Biochemistry
contact.country: USA
contact.institute: University of Georgia
organism: Pyrococcus furiosus DSM 3638
description: Glass slide, see protocol above.
manufacture_protocol: Each of the known 2192 ORFs in P. furiosus were amplified individually by PCR. Primers were selected to produce full-length PCR products. In case of large ORFs >2 kb PCR primers were designed to yield a PCR product around 1 kb. Expected PCR products with high homology to more than one locus were replace by a 50-mer specific oligo. Oligos were purchased from MWG Biotech (High Point, NC) in a 96-well format. Aliquots of the primers were used in 96-well format PCR reactions. PCR products were verified by gel electrophoreses. PCR products were subsequently purified using a 96-well PCR purification kit (Stratagene). Eluted DNA was mixed with 50% DMSO to minimize evaporation during arraying.
  The probes (PCR products) were be spotted onto amino silane coated glass slides (Asper or PerkinElmer) and cross-linked to the slides by UV irradiation. The microarrays were made in house at the UGA using a robotic slide printer (Omnigrid, GeneMachines) with Stealth SMP3 pins (Telechem). Arrays were spotted in triplicate in 8 subarrays (4 x 2), 19 x 19 spots with a 225 um spacing.