title: NLI_SSC_11.5K_cDNA_V1
status: Public on Dec 17 2007
contact.city: Beijing
contact.first: Jin
contact.last: Zhang
contact.email: zhangjin7688@163.com
contact.address: 2#, Yuanmingyuan West Road
contact.country: China
contact.institute: China Agricultural University
organism: Sus scrofa
manufacture_protocol: Plasmid inserts were amplified by PCR using T3 (5’-GTA AAACGACGGCCAGT-3’) and T7 (5’-CAGGAAACAG CTATGAC-3’) flanking vector primers in 50ul volume reactions. Amplifications were performed in a thermal cycler (PE Amp9700, Perkin-Elmer, USA). PCR reaction mixes contained 5ul 10×Taq polymerase buffer solution, 50 pmol of each primer, 4ul 10 mM dNTPs, and 1 U Taq polymerase. An initial 5 min denaturation at 95oC was followed by 35 cycles of 1 min denaturation at 95oC, 1 min annealing at 55oC and 1 min elongation at 72oC. The reaction ended with an additional incubation of 5 min at 72oC. PCR products were analyzed on 1.0% agarose gels. Samples containing multiple bands or weak products were reamplified. PCR products were precipitated with isopropanol and redissolved in 50% dimethyl sulphoxide (DMSO). A commercial OmniGridTM microarrayer (Genomic Instrumentation Services, Inc, San Carlos, CA) was employed to print all spots on Telechem Superamine slides (Arrayit, Sunnyvale, CA) and to verify reproducibility each spot was arrayed three replicates. Control samples included the expressed pig genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L24 (RPL24), eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), glutathione peroxidase 1 (GPX1), and peptidylprolyl isomerase A (cyclophilin A) (PPIA). Additional, control samples including three different Arabidopsis thaliana DNA fragments (GenBank accession numbers AC004146, AC007661 and U09332) and 12 spots of DMSO were also arrayed. The centre-to-centre distance between two adjacent spots was 225 m. After printing, the slides were baked for 1 h at 80oC and stored dry at room temperature until use.