title: ISU_Minion_Mhyo_IG_v1.0
status: Public on Jun 30 2010
contact.city: Ames
contact.first: Chris
contact.last: Minion
contact.email: fcminion@iastate.edu
contact.state: IA
contact.address: 1130 Vet Med
contact.department: VMPM
contact.country: USA
contact.institute: Iowa State University
contact.phone: 515-294-6347
organism: Mesomycoplasma hyopneumoniae
description: Corning UltraGAPS substrates (catalog #40016)
manufacture_protocol: Probes were designed for the intergenic regions of Mycoplasma hyopneumoniae. Intergenic regions were defined as noncoding DNA sequences between annotated genes in the M. hyopneumoniae genome sequence (GenBank accession # AE017332). Intergenic regions larger than 50 bp were considered for analysis, and those between 125-450 bp were considered for PCR-based probes. PCR primers were designed using the Primer3 online software program. PCRs were performed in 96-well plate format in a MJ Research Dyad thermocycler (Bio-Rad Laboratories, Inc. Waltham, MA) in 100 μl reactions using the following conditions: 1X MgCl2-free PCR buffer (Invitrogen); 3.5 mM MgCl2; 0.2 mM each dATP, dTTP, dGTP, and dCTP (NEB); 200 pmol of each primer (IDT); 2 units Taq polymerase (Invitrogen); and 100 ng M. hyopneumoniae strain 232 chromosomal DNA obtained via phenol-chloroform extraction. Thermocycler conditions started with a denaturation at 95ºC for 5 minutes followed by 30 cycles of denaturation at 94ºC for 1 minute, annealing at 56ºC for 1 minute, and elongation at 68ºC for 30 seconds and ending with a final elongation at 68ºC for 10 minutes. Products were confirmed by 1.2% agarose gel electrophoresis and purified via Mobio’s UltraClean PCR Clean-Up Kit according to manufactures protocol. Purified PCR products were quantified by visual inspection, dried via vacuum centrifugation, and resuspended to approximately 200 ng per μl in Corning spotting buffer (Corning). Products suspended in spotting buffer were electrophoresis in 1.2% agarose gels to confirm purity and concentration of each product. The PCR product microarrays were spotted to Corning UltraGAPS glass substrates in triplicate by the University of Iowa DNA facility.