title: Nitrosomonas europaea, whole genome microarray
status: Public on Mar 23 2006
contact.city: Riverside
contact.first: Lisa
contact.middle: Yael
contact.last: Stein
contact.email: lisa.stein@ucr.edu
contact.state: CA
contact.address: Geology 2207, 900 Univ. Ave.
contact.department: Environ. Sciences
contact.country: USA
contact.web_link: http://www.envisci.ucr.edu/index.php?file=faculty/stein/stein.html
contact.institute: University of California
contact.phone: 951-827-2704
organism: Nitrosomonas europaea
manufacture_protocol: Construction of whole open reading frame (ORF) microarrays for N. europaea was done by generating gene-specific fragments (<75% homology) by PCR using individually designed primer sets using PRIMEGENS software. All amplified products (8-16 reactions per primer set x 100 μl) were pooled together and purified using a robot (Biomek F/X Automated Workstation; Beckman). The range of product sizes was 100-1200 bp with most between 500-600 bp. Of the 2603 total predicted genes, 2318 ORFs were correctly amplified and specific 50-mer oligonucleotide probes (74 ORFs) were synthesized for the remainder, representing 96.8% of the total predicted gene content of N. europaea. Diluted PCR products (50 ng μl-1) were spotted onto CMT-GAPS slides (Corning, Corning, NY) using a BioRobotics Microgrid II microarrayer (Genomic Solutions, Ann Arbor, MI).