title: UCD_Ronald_XOarray_5K_V2.0
status: Public on Jun 14 2008
contact.city: Davis
contact.first: Malinee
contact.last: Sriariyanun
contact.email: macintous@gmail.com
contact.state: CA
contact.address: 451 East Health Sciences Drive
contact.department: Plant Pathology
contact.country: USA
contact.web_link: http://indica.ucdavis.edu/
contact.institute: University of California, Davis
contact.phone: 530-574-6568
organism: Xanthomonas oryzae pv. oryzae KACC 10331
  Xanthomonas oryzae pv. oryzicola BLS256
manufacture_protocol: Oligo Synthesis
  Oligos used for spotting were commercially synthesized by IDT (http://www.idtdna.com). As a control, an oligo was designed by Picky to detect hph but not Xo genes. This potential spiking control was included in 2 well positions on each 384-well oligo synthesis plate except last plate with one hph position. Because the positions of this spiking control were randomized across plates, this oligo also serves as check for array printing.
  Spotting
  All work at ArrayCore is performed in a HEPA-filtered clean room environment to minimize contamination from airborne particles. Oligonucleotides were suspended in 1x Nexterion Spot solution at a final concentration of 20 µM and spotted onto epoxy-coated glass slides (Schott-Nexterion, USA). Oligonucleotides were spotted using a Lucidea Array Spotter (Amersham) in a humidity controlled spotting chamber (70%) at room temperature. Microarrays were deposited using 190 µm column and row pitches, and spot diameters averaged 80 µm under these conditions. After spotting, slides were allowed to sit at 70% humidity overnight at room temperature to maximize oligo binding. Microarrays were allowed to dry at ambient conditions and stored in the dark under Argon at room temperature until use. Slides and spotting plates were tracked using the array spotter’s built in barcode reader and the information was used to generate the gene array layout file of the spotted 5k Xo oligonucleotide microarray.
  Amino-blocking Pre-treatment
  Prior to hybridization process, oligos-spotted slides were pretreated with blocking step to remove unbound DNA-molecules and buffer substances from the slides by extensive washing to avoid any interference with subsequent hybridization experiments. Spotted slides were incubated in amino blocking solution (5 g succinic anhydride in 315 ml n-methylpyrrolidone, 35 ml 0.2 M sodium borate ph 8.0) at room temperature for 15 mins. Slides were placed in 0.1% SDS solution 20 sec and washed with nanopure water 20 sec 2 timesand then were transferred into the sodium borohydride block solution.
  Sodium Borohydride Pre-treatment.
  To minimize non-specific autofluorescence from the spotted material , slides were placed into a block solution containing 2x SSC/0.05% SDS/0.25% NaBH4 (Biochemical Technologies, USA) and incubated at 42°C for 20 min. Slides were transferred to 1x SSC for 5 min at room temperature and then sequentially washed with vigorous stirring using fresh 1x SSC (3 × 5 min, room temperature), 0.2x SSC (4 × 2 min, room temperature), and Nanopure water (1 × 2 min, room temperature). Slides were spin-dried (1000 rpm, 10 min) and stored under Argon until use.