title: KARC Schizosaccharomyces pombe CEN array
status: Public on Dec 01 2007
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 534 spots included: 514 spots which correspond to the sense or antisense strands of 257 sequences (ura4, GFP and 255 sequences from cen regions), and 20 negative control spots. ID number 50 and 306 are assigned to the #50 probes that were not included in the array (see the Manufacture Protocol).
manufacture_protocol: 255 probe sequences from centromere regions were generated by PCR. 173 of these sequences occurred between lys1 to the right edge of cntI. 56 sequences were from cen2, and the remaining 26 sequences from cen3. Probes for cen2 and cen3 were selected only from specific regions unique to each centromere. #50 probes of cen1 were not included in the array because they were not amplified by PCR. To make specific probes for each strand of the sequence, two primer sets were prepared for each sequence. In one set, the 5' end of the forward primer was modified with an amino group. In the other set, the 5' end of the reverse primer was modified with an amino group. A GeneAmp PCR System 9700 (Applied Biosystems) and z-Taq polymerase (TAKARA) were used for PCR. PCR conditions were as follows: 40 cycles of denaturation at 95°C, extension at 72°C, and annealing, depending on the Tm values of the primers, at 54-63°C. The PCR products were purified by using Multiscreen96 (Millipore) or NucleoFast96 (MACHEREY-NAGEL) kits and their concentrations were measured using a SpectraMax190 (Molecular Devices).
  For each probe, 4µg of probe DNA was dissolved in 20µl spotting buffer (150mM Na-phosphate buffer pH8.5).These probes were spotted on a Surmodics 3D-Link Slide by a PixSys4500 (Genomic Solutions) to produce the centromere array. Spotting was carried out by the NippnTecnoCrasta e-Array system. 534 spots were applied to each cen array. The diameter of each spot was 380µm and the pitch 920µm. To covalently bind probe DNA to the glass slide, a blocking procedure was carried out as follows: the stamped slides were incubated in a humidity chamber (~75% humidity) for 2 days, and then were soaked in a pre-warmed blocking buffer (50mM ethanolamine, 0.1M Tris-Cl pH9, 0.1% SDS) at 50°C for 15-30 minutes. The slides were rinsed twice with distilled water (DW), soaked in a wash buffer (4xSSC, 0.1% SDS) at 50°C for 15-60 minutes, and again rinsed twice with DW. To remove the unmodified anti sense strand of the probe DNA, the slides were boiled in DW for 2 min. After rinsing twice in DW, the slides were dried.