title: USITB Escherichia coli W3110 9.2k array v 1.0
status: Public on Sep 23 2008
contact.city: Stuttgart
contact.first: Karin
contact.last: Lemuth
contact.address: Allmandring 31
contact.country: Germany
contact.web_link: http://www.ibvt.uni-stuttgart.de/index.html
contact.institute: Universität Stuttgart
organism: Escherichia coli str. K-12 substr. W3110
description: epoxy coated
manufacture_protocol: Whole genome E. coli K12 DNA-microarrays were printed using the Microgrid II robot of Biorobotics (now Genomic Solutions, Huntingdon, United Kingdom) by spotting oligonucleotides (50mer, dissolved in buffer A; E. coli K-12 V2 Oligo set from MWG, Ebersberg, Germany) representing all E. coli K-12 ORFs on epoxy-coated glass slides (MWG) as duplicates. Oligonucleotides were delivered in 12 384 polypropylene microtiter plates with F-bottom (Greiner, Frickenhausen, Germany). For details on probe design please see the manufacturer’s protocol (http://ecom2.mwgdna.com/services/array/download.tcl). Tungsten microarray split pins (PT 3000 – Point Technologies, Inc., Colorado, USA) were used for spotting. 27 slides were manufactured per printing process. 3 slides were discarded as so called “pre-spot slides”, 24 slides were used for microarray experiments.
  Printing was performed using the following conditions (Software: TAS Application Suite V. 2.6.0.2):
  •	Tool: 4x4 configuration
  •	Group (Tool Type): 2. Micro Spot (384 Well)
  •	Source Action: dwell/remove one lid at a time
  •	60 spots / source visit, 10 of them were pre spots
  •	soft touch distance: 1.5 mm
  •	humidity control: minimal: 58%; target humidity: 63%
  •	washing conditions: 3 x at the beginning, 5 x at the end, bath 1 two seconds, bath 2 three seconds (2 mm wiggle); washing solution: ddH2O.
  •	all replikates were printed on the first slide first.
  After printing, microarrays were incubated in a hybridization oven (Whatman Biometra) at 42°C over night together with a ddH2O filled petri dish covered with looped aluminium foil. Afterwards, microarrays were washed stirring constantly with 0.2 % SDS solution (2 min), two times in ddH2O (1 min) at room temperature and afterwards incubated in the hybridization oven (Whatman Biometra) for 20 min in ddH2O at 50°C. Microarrays were nitrogen dried and stored in a dark and dry place until hybridization.
  Quality control: staining with SYBR SafeTM DNA Gel Stain (MolecularProbes) by incubation of the printed area with 750 µL of the solution and incubation for 3 min at room temperature. Arrays were washed four times with 0.5 x TBE-Puffer (5 x stock solution: Tris-Borat 0.45 M; EDTA 0.01 M) and scanned afterwards.
  For data analysis, a “gal file” was produced using the software TAS application suite version 2.6.0.2 from Genomic Solutions.