title: Rhodopseudomonas palustris whole genome microarray
status: Public on Nov 10 2005
contact.city: Seattle
contact.first: Yasuhiro
contact.last: Oda
contact.email: yasuhiro@uw.edu
contact.state: WA
contact.address: Box 357242, 1705 N.E. Pacific Street, HSB K-354
contact.department: Department of Microbiology
contact.country: USA
contact.institute: University of Washington
organism: Rhodopseudomonas palustris
manufacture_protocol: PCR primers for 4508 out of 4836 predicted ORFs of the Rhodopseudomonas palustris chromosome were designed using the PRIMEGENS software. Each gene was amplified 16 times in parallel 100 µl reactions in 96 well plates. All of the amplified products were pooled and purified using a Biomek F/X Automated Workstation (Beckman). All the amplified products were analyzed on 1.5% agarose gels, and were considered correct if the PCR reactions contained a single product of the expected size. Of the 4508 total predicted genes, 4350 ORFs were correctly amplified, which represents approximately 90% of the genome. Specific 50-mer oligonucleotide probes (385 ORFs) were synthesized for the genes that were not amplified. In total, the PCR amplicons and oligo probes represented 98% of the total predicted gene content of Rhodopseudomonas palustris. The purified PCR products were diluted in 50% DMSO to a minimum concentration of 50 ng/µl. Probes were then printed onto Corning UltraGAPS coated slides (Corning, Corning, NY) with a BioRobotics Microgrid II printer (Genomic Solutions, Ann Arbor, MI) as recommended by the manufacturer, and were UV cross-linked to the surfaces of slides (Statagene UV Crosslinker; Stratagene, La Jolla, CA). Each slide was printed with probes representing two copies of the genome.