title: Solanum tuberosum 12.3K RLP array Version IV
status: Public on Mar 06 2010
contact.city: Seibersdorf
contact.first: Friederike
contact.last: Trognitz
contact.email: friederike.trognitz@ait.ac.at
contact.state: NÖ
contact.address: Seibersdorf
contact.department: Health & Environment
contact.country: Austria
contact.institute: AIT Austrian Institute of Technology GmbH
contact.phone: 43(0)50550-3528
organism: Solanum tuberosum
manufacture_protocol: The microarray was based on an enriched, full-length cDNA library of S. tuberosum var. Yungay, which had been challenged with Phytophthora infestans complex race for four days. The library was made by VERTIS GmbH (Freising, Germany). The cDNA was inserted to the EcoRI/NotI digestion site using vector pBS IIsk+ and transformed into TransforMax TM Ec100TM-T1 electrocompetent cells. Plasmid DNAs of 3840 clones were obtained by adding a small portion of bacterial culture into TempliPhi DNA Sequencing Template kit (GE Healthcare) according to the manufacturer’s instructions. Inserts were amplified by PCR in 50 µl reactions containing 0.5 µl of a 1:100 dilution of the TempliPhi, 5mM dNTPs (Invitrogen, Oslo, Norway), 2.5 mM MgCl2, 1x BD buffer (Solis Biodyne), 0.5 µM each of primers T7 and T5, and 2 U of Firepol polymerase (Solis Biodyne, Tartu, Estonia). The amplification was done in a GeneAmp PCR System 2700 thermocycler (Applied Biosystems) under the conditions: 95°C for 1 min, 2 min at 50°C and 2 min at 72°C and 30 cycles consisting of 95°C 1min, 56°C 2 min and 72°C for 1 min and a final elongation step at 72°C for 5 min.
  In addition to the Yungay cDNA library, another 1173 cDNA clones were ordered from the University of Arizona Genomics Institute corresponding to stress-related genes and treated as described above and amplified with the corresponding primers. Furthermore, nine housekeeping genes were included. Genes and primers are listed in the array file. They were amplified by using 1 µl of cDNA from S. tuberosum var. Yungay in a mix containing 2.5 mM MgCl2, 0.25 mM dNTPs (Invitrogen, Oslo, Norway), 0.5 µM of each primer, 1x BD buffer (80mM Tris•HCl, 20 mM (NH4)2SO4) (Solis Biodyne, Tartu, Estonia) and 0.2 µl FirePol (Solis Biodyne, Tartu, Estonia) in a PCR reaction of 95°C for 1 min followed by 35 cycles with 95°C 1 min, 55°C 1 min, 72°C 1 min and a final elongation step for 10 min at 72°C.
  Twenty µls of all amplicons were lyophilized and then dissolved in 10 µl spotting buffer (3xSSC and 1.5 M betaine). Probes were spotted in duplicate with an Omnigrid 100 (GeneMachines) equipped with 48 TeleChem pins on CAPS II coated slides (Corning) in a 200 µM spot distance and 40% humidity.