title: UWVJLAB RT 150 v.1.0
status: Public on Jul 01 2006
contact.city: Woods Hole
contact.first: Neelakanteswar
contact.last: Aluru
contact.email: naluru@whoi.edu
contact.state: MA
contact.address: 45 Water Street
contact.department: Biology
contact.country: USA
contact.institute: Woods Hole Oceanographic Institution
contact.phone: 508-289-3607
organism: Oncorhynchus mykiss
manufacture_protocol: PCR products were cloned into the Invitrogen pCR-Topo II vector (Invitrogen, Burlington, ON, Canada) according to the manufacturer’s protocol.  Ligation of the PCR products into the vector was verified by PCR with the vector specific M-13 forward and reverse primers.  Plasmids containing the desired PCR fragments were isolated using a Qiaprep Spin miniprep plasmid isolation kit (QIAgen).  All plasmids were sequenced, for correct insert confirmation, at the Center for Environmental Health, at the University of Victoria (Victoria, B.C. Canada).
  
  Each gene fragment was amplified in ten PCR reactions (100 µl each or 1 ml total) in a thermal cycler (MJ Research, Watertown, MA, USA) using gene specific primers in the following PCR reaction: 1X Taq Polymerase buffer containing magnesium chloride, each dNTP at a concentration of 300 µM, 3.75 U of Taq (Promega, Madison, WI, USA), 3 ng of plasmid, and 0.6 µM of each specific primer.  The following conditions applied: 35 cycles of 94°C for 30 s, appropriate annealing temperature (45 to 55 oC depending on gene specific primers) for 60 s, and 72 °C for 2 min followed by a final 12 min extension at 72°C.  PCR products were visualized on a 1.2% agarose gel stained with 1 µg of ethidium bromide/ml to confirm correct size of amplicon as well as minimal primer dimer.  The ten PCR reactions for each gene fragment were pooled and purified using the Qiagen QIAquick PCR Purification kit.  Concentration of each gene fragment was determined via spectrophotometry, the samples were speed vacuum dried, resuspended in 3X SSC to a final concentration of 0.5 µg/ml for each gene fragment.  The gene fragment was confirmed with a 1.2% agarose gel.
  
  Prior to printing, cDNA clones were diluted to 50 ng/µL in 3x SSC and placed in a 384 well plate.  The cDNA fragments were printed on ArrayIt Superamine glass slides (Telechem, Sunnyvale, CA, USA) using a BioRad ChipWriter Pro robotic System (BioRad, Mississauga, ON, Canada) equipped with 4 pins for printing one array (Telechem).  Each cDNA fragment was printed side by side in triplicate.  In addition, a 500 bp fragment from the Lambda Q bacterial gene was printed onto each slide as a control for hybridization inefficiencies.