title: KARC Schizosaccharomyces pombe 5K Ver. D ORF array
status: Public on Mar 20 2006
contact.city: Kobe
contact.first: Atsushi
contact.last: Matsuda
contact.address: 588-2, Iwaoka, Iwaoka-cho, Nishi-ku
contact.country: Japan
contact.institute: National Institute of Information and Communications Technology
organism: Schizosaccharomyces pombe
description: The breakdown of 5184 spots is as follows; 4929 fission yeast genes (cdc2, nuc1, and nda3 are excluded) are spotted once on a slide, cdc2, nuc1, nda3, and GFP are spotted once for each block (16 in total). Representative values of r' for these 4 genes were average of their 16 spots. ID numbers from 5185 to 5188 are assigned to their representative values. 11 negative control spots are common to each block (A to P except, 176 in total). The reaction mixture except primers was spotted at negative control spots. Only spotting buffer were spotted at 8 blank spots. Spots for the following 7 ORF, SPAC212.10, SPAC3G9.16c, SPBC19G7.12c, SPCC784.05c, SPCC1442.04c, SPAC8E11.04c, SPBC2A9.08c, are ignored in data processing (see details in http://www-karc.nict.go.jp/d332/CellMagic/index.html).
manufacture_protocol: Probe DNA used for the microarray was generated by PCR from the code region of each gene that has been described in annotated fission yeast genome sequences(Wood et al., 2002 Nature 415, 871-880). The continuous 300bp+/- 10bp exon sequence nearest to the 3'-end of the gene was selected as a probe. When the longest exon of the gene is shorter than 290bp, the longest exon was used as a probe. The primer used for PCR was designed by using the primer design supporting tool, Primer3(http:://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). The length of each primer is 30+/-3bp. Tm of the primer is 60-70C. The forward primers are modified by the amino group at their 5' end. Wild type L972 strain's genome DNA was used as the temperate of the first rounds of PCR. For the second and later round of PCR, the PCR products of the first round of PCR were used as a temperate. GeneAmp PCR System9700(Applied Biosystemes) and z-Taq polymerase(Takara) were used for PCR. PCR was done as follows: 40 cycles, 95C for denature, 72C for extension, for annealing 54-63C according to the Tm value of primers.
  The PCR product was purified by using Multiscreen96(Millipore) or NucleoFast96(MACHEREY-NAGEL) and measured the density by using SpectraMax190(Molecular Devices). A part of the product was confirmed by agarose gel electrophoresis. The dried up probe DNA 4 micro g is dissolved in 20 microl spotting buffer (150mM Na-phosphate buffer pH8.5). Probe DNA was stamped on Surmodics 3D-Link Slide by GTMASS STAMPER (Nippon Laser Electronics). 16 pins of GTMASS STAMPER stamp 324(18x18) times respectively on a slide to form 16 blocks (A to P) including 5184(16x18x18) spots in total. The diameter of each spot is about 150 microm, and the pitch is 190microm. The stamped slides are incubated in a humidity chamber (about 75% in humidity) for 2days, then are soaked in a pre-warmed blocking buffer (50mM ethanolamine, 0.1M Tris-Cl pH9, 0.1% SDS) at 50C, for 15-30 minutes. The slides were rinsed twice with distilled water (DW), and soaked in a wash buffer (4xSSC, 0.1% SDS) at 50C, for 15-60 minutes. then, are rinsed twice with DW, and boiled in DW for 2 minutes. After rinsing twice in DW, the slides were dried up.