title: UMC_VET Canis familiaris 21K cDNA array
status: Public on Apr 25 2007
contact.city: Utrecht
contact.first: Nagesha
contact.middle: A S
contact.last: Rao
contact.email: n.rao@ncmls.ru.nl
contact.state: Utrecht
contact.address: Yalelaan104
contact.department: Veterinary sciences
contact.country: Netherlands
contact.web_link: http://www.vet.uu.nl/viavet
contact.institute: Utrecht University
contact.phone: 31-30-2531709
organism: Canis lupus familiaris
description: Slide Dimensions (LxWxH):  	 25.3x75.5x1.1 mm
manufacture_protocol: A canine specific collection of 20,160 non-redundant clones of 3’-UTR cDNA fragments inserted in pBluescript II KS and cloned into E Coli DH10B were purchased from Lion Bioscience GmbH (Heidelberg, Germany). Individual clones were grown overnight in LB supplemented with 50 µg/ml ampicillin and heated at 950C for 10 minutes. Four µl of bacterial culture was subsequently used for PCR amplification of the clone inserts using plasmid specific primers sense 5’-AGCGTGGTCGCGGCCGAGGT and antisense 5’-TCGAGCGGCCGCCCGGGCAGGT and 35 cycles of denaturation (940C, 30 sec), annealing (660C, 30 sec) and extension (720C, 1 min) using standard PCR protocols. Hundred µl PCRs were typically performed in triplicate for each clone in order to obtain enough PCR product for microarray production. Each PCR product was checked for contaminations using gel electrophoresis, and flagged if no or more than one PCR product was detected. PCR products were purified using 96-well MultiScreen plates with FB filters according to the manufacturers protocol (Millipore, Amsterdam, The Netherlands). After measurement of the concentration PCR products were air dried and reconstituted to a final concentration of 150 ng/µl in print buffer (150 mM phosphate buffer pH 8.5), and 10µl of each product was transferred to 384 well plates. PCR products that did not reach the desired concentration were flagged. PCR products were spotted on UltraGAPS slides (Corning) using the MicroGRID II arrayer (Genomic Solutions, Huntingdon, UK), air dried and immobilized by UV cross linking using a Stratalinker2400 (Stratagene, La Jolla, CA) for 10 min at 2400 mJ.  Microarrays were stored at room temperature in desiccators until needed.