title: Maize SAM1.2 Array
status: Public on Nov 07 2006
contact.city: Ames
contact.first: Patrick
contact.middle: S.
contact.last: Schnable
contact.email: schnable@iastate.edu
contact.state: IA
contact.address: 2035B Roy J Carver Co-Lab
contact.country: USA
contact.institute: Iowa State University
contact.phone: 515-294-0975
organism: Zea mays
manufacture_protocol: A 15,680 element microarray chip (SAM1.2 chip) was generated at Iowa State University’s Center for Plant Genomics.
  Of the 15,680 elements printed on the UltraGAPs slide (Corning, Inc.), 5,753 cDNA clones from the Stanford UnigeneI developed by the National Science Foundation-funded Maize Gene Discovery Project (http://www.maizegdb.org) and re-sequenced at Dr. Schnable’s lab, 1,058 cDNA clones from the UnigeneIV-1091, 6,711 cDNA clones from the library 3529, 1,530 cDNA clones from the ISUM3, ISUM4, ISUM5, ISUM6, ISUM7, UGA-ZmSAM-XZ1 and UGA-ZmSAM-XZ2 libraries (http://schnablelab.plantgenomics.iastate.edu/research/genomics/htp_est/) prepared at Iowa State University as part of a National Science Foundation Plant Genome Project; In addition, 192 human gene spots are also included on the chip.
  The full-length inserts from the library 3529 were PCR amplified from denatured O/N E. coli culture, then purified and quantified (Nakazono et al. 2003 Plant Cell, 583-596). The rest ESTs were PCR amplified using plasmid DNAs as template in 100 µl PCR reactions containing 1X PCR buffer (200 mM Tris-HCl, pH 8.4 and 500 mM KCl), 2.5 mM MgCl2, 0.2 mM dNTPs, 0.2 µM of each primer, and Taq polymerase. The PCR program cycle was: 94°C for 3 min followed by 35 cycles of 94°C for 45 sec, 60°C for 45 sec, 72°C for 2 min 30 sec, and a final elongation step of 2 min. Most PCR negative (no band, multiple bands or smears) wells were removed, and a few were spotted onto the slides with labeling badPCR in the GeneID file. PCR products were purified using Millipore 96-well multiscreen filter plates (LSKC09601) as recommended by the manufacturer and eluted in 50 µl of water. Five microliters of each purified PCR product were electrophoresed on a 1% agarose gel to examine quantity quality. The remaining 45 µl of purified PCR product were dried to completion in a vacuum oven and resuspended in 50% DMSO with a final DNA concentration 300 to 500 ng/µl.
  The PCR amplified cDNA inserts were printed on UltraGAPS, which is uniform covalently bound of pure gamma amino propyl silane coating (Corning, NY, Cat. No. 40015).  The whole array consists of 48 subgrids printed in 18- x 54-mm array area.
  PixSys 5500 arrayer (Cartesian Technologies, Irvine, CA) equipped with ChipMaker3 pins (TeleChem, Santa Clara, CA) is used for printing. Slides were UV cross-linking at 300 mJ with a Stratalinker (Stratagene) for immobilization, and stored in desiccator at ambient temperature.