title: VU University Orchesella cincta array 7K
status: Public on Apr 01 2009
contact.city: Amsterdam
contact.first: Dick
contact.last: Roelofs
contact.email: dick.roelofs@falw.vu.nl
contact.state: Noord-Holland
contact.address: De Boelelaan 1085
contact.department: Animal Ecology
contact.country: Netherlands
contact.web_link: http://www.falw.vu.nl/nl/onderzoek/ecological-sciences/animal-ecology/staff/dick-roelofs.asp
contact.institute: VU University
contact.phone: +31-20-5987078
organism: Orchesella cincta
description: hydrophilic polymer containing N-hydroxysuccinimide (NHS) ester reactive groups
manufacture_protocol: We amplified cDNAs from our previously obtained cadmium exposed SSH library together with clones derived from a cDNA library of cadmium-exposed gut tissues with amine-linked primers. Amine-tagged cDNAs were spotted onto Codelink Activated slides (GE Healthcare). Each cDNA was printed twice on one array and two arrays were printed on one slide, so that each cDNA was four times technically replicated on a slide. Pins of the printer resampled the cDNAs before printing the replicate array, so each slide contains two separately printed arrays.
  Printing procedure:
  The amine-labeled PCR products (0.1–1 kb)were diluted at a concentration of 0.1–0.5 mg/ml DNA in printing buffer (50 mM sodium phosphate, pH 8.5).
  Printing occured in an environment with relative humidity < 50% to
  retain binding activity.
  DNA was printed onto the Codelink activated slides to produce microarrays on a Biorobotics MicroGrid arrayer
  Printed slides were placed into a slide storage box in the saturated NaCl chamber.
  chamber was sealed and incubated overnight at room temperature
  residual reactive groups were blocked using pre-warmed blocking solution
  (0.1 M Tris/0.1 % SDS, 50 mM ethanolamine, pH 9.0)at 50 °C for 30 min.
  slides were rinsed twice with deionized water.
  slides were washed with 4X SSC / 0.1% SDS (pre-warmed to 50 °C)
  for 30 min on the shaker. At least 10 ml per slide was used.
  Slides were placed into boiling water
  for 2 minutes to denature double stranded DNA
  slides were rinsed twice with deionized water.