title: RIKEN-LNCD-GSL_Mouse_16k_1.0
status: Public on Jul 29 2008
contact.city: Wako
contact.first: Charles
contact.last: Plessy
contact.email: plessy@riken.jp
contact.state: Saitama
contact.address: 2-1 Hirosawa
contact.department: DRI
contact.country: Japan
contact.institute: RIKEN
contact.phone: +81-48-467-9237
organism: Mus musculus
description: This platform was prepared jointly by the Laboratory for Neuronal Circuit Development (LNCD), RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan, and the Genome Science Laboratory (GSL), Discovery and Research Institute, RIKEN Wako Institute, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
manufacture_protocol: cDNA clones were rearrayed with a Q-bot instrument (Genetix) from 20 % glycerol/LB ampicillin stocks that were kept at -80 °C. A growth check was performed on all glycerol stocks. For the growth check 384-well plates were translated into four 96-well plates per 384-well plate and one bacterial culture per glycerol stock was inoculated. All plates were analyzed after 18 h of growth at 37 °C by visual inspection. We performed one Plasmid DNA Preparation per Glycerol Stock using a Montage Purification Kit (Millipore) and our robotic systems. One quality control was performed on three clones per plate, and the yield was 2.66 µg DNA ± 1.73. For the preparation of PCR templates, we diluted the plasmid DNA preparation by 1:500. 5 µl of template were amplified in 50 µl reactions in 96-well plates with 1.25 U Expand High Fidelity Taq/Tgo polymerase blend (Roche), 15 pmol of forward primer (tgtaaaacgacggccagt), 15 pmol of reverse primer (agcggataacaatttcacacagga) and 200 µM of each dNTP (TaKaRa) in 1 × Expand High Fidelity buffer (Roche). The program of the thermocycler was 2 min at 94 °C followed by 30 cycles of 15 s at 94 °C, 30 s at 60 °C, and 2 min 30 s at 68 °C, plus a final step of 7 min at 72 °C. The amplification products were then purified with Multiscreen PCR plates (Millipore) following manufacturer instruction, and dried. With this protocol, we could achieve an average yield of > 8 μg for 92.7 % of the wells.
  The PCR-amplified cDNAs were resuspended in 50 % DMSO (Nacalai Tesque, Kyoto, Japan), and microarrays were printed on UltraGAPS slides (Corning) using an OmniGrid arrayer (Genomic Solutions) with Microarray Stealth Spotting pins (SMP3B, TeleChem).