title: NRC-BRI_Hydrocarbon degrading genes v6.1_141
status: Public on Aug 06 2009
contact.city: Montreal
contact.first: Etienne
contact.last: Yergeau
contact.email: Etienne.Yergeau@cnrc-nrc.gc.ca
contact.state: Qc
contact.address: 6100 Royalmount Ave
contact.department: Environmental Microbiology
contact.country: Canada
contact.institute: NRC-BRI
organism: Bacteria
  Archaea
manufacture_protocol: All PCR amplified target fragments were produced using the rTaq DNA polymerase (Amersham Bioscience, Baie d’Urfé, QC, Canada) with the appropriate DNA template (either genomic DNA or plasmid) and primers.  The amplification program was as follows: hot start at 96°C for 5 min; 30 PCR cycles consisting of denaturation at 94°C for 1 min, annealing at the appropriate temperature (variable for each PCR amplified fragment) for 1 min and elongation at 72°C for 1 min; final elongation at 72°C for 3 min.  Each PCR amplified fragment was purified using the Qiaquick PCR Purification kit (Qiagen Inc, Mississauga, ON, Canada) according to the manufacturer’s protocol with the exception that the DNA fragment was eluted from the column with H2O freshly brought to pH 8.0 with 2N NaOH. Each fragment was dried and re-dissolved to a final concentration of 200 ng/µL in 50% DMSO (Sigma, St. Louis, Mo) for printing.  In order to monitor for printing problems, a 500 base pair fragment amplified from lambda DNA was added as a printing control (PC) to each target fragment at a concentration of 20 ng/µL.  The targets were arrayed with four Stealth Micro Spotting SMP3 pins (TeleChem Intenational Inc., Sunnyvale, CA) at a spacing of 250 µm on GAPS II microarray slides (Corning Inc, Acton, MA) using a VersArray ChipWriter Pro system (Bio-Rad, Hercules, CA).  The DNA target fragments were immobilized by UV cross-linking at 300 mJ in a UV Stratalinker 1800 (Stratagene,  La Jolla, CA) and then baked overnight at 80°C.