title: GRASP 16K version 2
status: Public on Aug 05 2005
contact.city: Victoria
contact.first: Ben
contact.middle: F
contact.last: Koop
contact.email: bkoop@uvic.ca
contact.state: BC
contact.address: PO Box 3020 STN CSC
contact.department: Biology
contact.country: Canada
contact.institute: The University of Victoria
contact.phone: (250) 472-4067
organism: Osmerus mordax
  Oncorhynchus mykiss
  Salmo salar
  Coregonus clupeaformis
  Oncorhynchus tshawytscha
description: The microarrays have a 4-by-12 metagrid layout with 19 X19 spot subgrid, each spot having an approximate diameter and pitch of 100 um and 0.20 mm, respectively. A 280 bp GFP (green fluorescent protein) cDNA was amplified from a GFP clone (Clontech) using the primers (5'-GAAACATTCTTGGACACAAATTGG-3') and (5'-GCAGCTGTTACAAACTCAAGAAGG-3') and printed in each subgrid corner to assist in gridding. Six exogenous genome (Arabidopsis) cDNAs were amplified from the following clones kindly provided by The Arabidopsis Information Resource:  rubisco activase [GenBank:T41667], protochlorophyllide reductase precursor [GenBank:R30630], chlorophyll a/b-binding protein CP29 [GenBank:N65746], PSII oxygen-evolving complex protein 2 [GenBank:H36167], tonoplast intrinsic protein root-specific RB7 [GenBank:AA067532] and ferredoxin (2Fe_2S) precursor [GenBank:W43249] and spotted in quadruplicate on each microarray. Also, a ubiquitin normalizer serially diluted (50 pg, 5 pg, 500 fg, 50 fg, 5 fg, 0.05 fg and 0.005 fg) was applied to the array. Spot morphology was assessed by visual inspection, SYBR® Green 1
  (Molecular Probes) staining or hybridization with labeled non-specific probe.
manufacture_protocol: Clones were robotically rearrayed from daughter glycerol stock 384-well plates into 96-well plates pre-filled with 7% glycerol in LB + ampicillin, incubated overnight at 37°C, and checked for uniform optical density. Plasmid inserts were PCR amplified in a Tetrad PTC-200 thermocycler (MJ Research) using 1 ul overnight culture, 0.2 mM M13/pUC forward primer (5'-CCCAGTCACGACGTTGTAAAACG-3'), 0.2 mM M13/pUC reverse primer (5'-AGCGGATAACAATTTCACACAGG-3'), 2 mM MgCl2, 10 mM Tris-HCl, 50 mM KCl, 250 mM dNTPs, 1U AmpliTaq (Perkin Elmer), and nuclease-free H2O (Gibco) to 100 ul. PCR conditions were: 2 min for 95°C; then 35 cycles of 95°C for 30 sec, 60°C for 45 sec, 72°C for 3 min; followed by 72°C for 7 min. Five ul of each PCR product were run on a 1% agarose gel to assess yield and quality. PCR products were robotically cleaned (Qiagen) and consolidated into 384-well plates, lyophilized by speed-Vac, and resuspended in 20 ul 3X SSC. Each purified PCR product concentration was determined and diluted to give a final concentration of 400ng/uL.
  All cDNAs were printed as single spots on EZ Rays aminosilane slides (Matrix/Apogent Discoveries) with the Biorobotics Microgrid II microarray printer (Genomic Solutions). Microspot™ 10K quill pins (Biorobotics) in a 48 pin tool were used to deposit approximately 0.5 nl (0.2 ng cDNA) per spot onto the slide. The slides were crosslinked in a UV Stratalinker 2400 (Stratagene) at 300 mJ.