title: GynObsLU Human 800 PE-associated cDNA
status: Public on Feb 28 2011
contact.city: Uppsala
contact.first: Katja
contact.last: Junus
contact.address: University Hospital
contact.department: Department of Women's and Children's Health
contact.country: Sweden
contact.institute: Uppsala University
organism: Homo sapiens
manufacture_protocol: cDNA clones were amplified using QuantiTaq™ polymerase and M13F / M13R primers as described by the manufacturer's protocol (GeneCopoeia, Germantown, USA).
  For each reaction, 5 μl plasmid templates were mixed with 1× PCR reaction buffer (Cat No. QU-02-10 GeneCopoeia), 0.2 mM dNTPs (Cat No. QT-04-250 GeneCopoeia), 1 μM of M13F / M13R primer respectively, 1.5mM MgCl2 and 1 U QuantiTaq polymerase (Cat No. QT-01-500 GeneCopoeia). PCR amplification was conducted with one cycle of 2 minutes of initial heating to 96°C, 29 cycles of denaturing at 94°C for 1 minute, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes, and one final cycle of holding at 72°C for 5 minutes. PCR products were purified using the NucleoFast 96 PCR Clean-Up Kit (ClonTech, Mountain View, USA). Briefly, PCR product was transferred to a NucleoFast 96 PCR plate and contaminants were filtered through by means of vacuum pressure. Membranes were washed once with 100 μl of nuclease-free water after which membranes were dried. Purified PCR product was recovered by adding 80μl recovery buffer and incubating the plates on shaker for 10 minutes. PCR products were verified on 1.2% agarose gels, 1.2g agarose, 100ml 1× TAE buffer (40mM tris pH 8.3, 1mM EDTA) and 6μl etidium bromide using 1× TAE as running buffer.
  Microarrays printed with our 800 cDNA-clones as well as an oligoset containing 27,000 oligos (OPERON v 2.1 human 70 mer oligo set, Operon, Huntsville, USA) were produced at the Swegene DNA Microarray Resource Center, Department of Oncology, Lund University, Sweden. Our 800 clones were printed in three replicates on each slide as a mean of quality control, allowing us to remove spots affected by local artifacts. Probes were dissolved in Corning Universal Spotting solution (Corning, Acton, USA) and printed on aminosilane coated glass slides (UltraGAPS, Cat. No. C40017, Corning) using a MicroGrid2 robot (BioRobotics, Cambridgeshire, UK) equipped with MicroSpot 10K pins (BioRobotics). Following printing, arrays were placed in a desiccator to dry for 48 hours, rehydrated for 1 second over steaming water, snap dried on a hot plate (98°C), and UV cross-linked (800mJ/cm2).