title: IGB_mouse_+33k_McC14
status: Public on Jan 31 2014
contact.city: Graz
contact.first: Andreas
contact.last: Prokesch
contact.email: andreas.prokesch@medunigraz.at
contact.state: Styria
contact.address: Harrachgasse 21/7
contact.department: Institute of Cell Biology, Histology, and Embryology
contact.country: Austria
contact.institute: Medical University Graz
organism: Mus musculus
manufacture_protocol: See publications: Hackl et al., Genome Biology,2005; Prokesch et al., Nucleic acid research, 2011; Pinent M et al., Genomics, 2008; Details:The mouse cDNA array platform containing 33184 spots was produced in-house. PCR amplification of several cDNA libraries (see(Hackl et al., 2005))  using universal primers (M13 Forward: 5’-GTT TTC CCA GTC ACG ACG TTG-3’, M13 Reverse: 5’-GAG CGG ATA ACA ATT TCA CAC AG-3’, end concentration: each 0.2 µM) was performed with a PTC 225 Tetrad (MJ Research) thermal cycler. The reaction volume of 100 µl contained 1x reaction buffer, 0.04 U/µl HotfirePol DNA Polymerase, 2mM MgCl2 (all from Solis Biodyne), dNTP mix (0.25mM each, Peqlab), primers and 2 µl template. After activation of the polymerase (95°C, 15 min) the program (95°C for 1 min, 62°C [decreasing to 52°C within the first 20 cycles] for 45 sec, 72°C for 2 min) was cycled 30 times, followed by a final elongation phase (72°C, 15 min). Products were visualized on 1.5% agarose gels (24x24 cm) with ethidium bromide. Plates with a low performance (high number of double bands or missing bands) were repeated. Unpurified PCR products were spotted in 3xSSC, 1.5M Betaine with a Microgrid 2 robot (Biorobotics) on epoxy silane slides (Schott) under stringent atmospheric conditions (21°C ± 1 and relative humidity 50% ± 5).