title: Institut Pasteur of Lille Cichorium intybus 1.5k cDNA array
status: Public on Apr 06 2009
contact.city: Villeneuve d'Ascq
contact.first: Anca
contact.last: Lucau
contact.email: Anca.Lucau@univ-lille1.fr
contact.address: Cité Scientifique
contact.department: Biology
contact.country: France
contact.institute: University Lille 1
contact.phone: (33)320434428
organism: Cichorium intybus
description: Ci2kcDNA
  The Platform data table is a condensed representation of the array.
  The full array layout is linked below as a supplementary file.
manufacture_protocol: Chicory annotated ESTs (Legrand et al, 2007), representing 1,098 unique genes, were used to produce microarrays. ESTs are integrated to the Compositae Genome Project library (http://compgenomics.ucdavis.edu/compositae_index.php).
  Six microliters of bacterial culture stored with glycerol were put into 100 µl TTE buffer (1% triton ×100, 20 mM Tris-HCl, 2 mM EDTA, pH 8.0) in 96 well plates. Plates were incubated 10 min at 95°C and spinned at 1,200 g for 5 min. The cDNA inserts were amplified using aminated adaptator-specific forward primer NP1 and reverse primer NP2 (both from Clontech) flanking the cDNA insert. For each clone, 3 independent reactions were carried out in a volume of 100 µl containing 1× ThermoPol Detergent Free Reaction Buffer (Biolabs), 2 mM MgCl2, 0.25 mM of each dNTP, 0.25 µM of each primer, 2.5 U of Taq DNA Polymerase (Biolabs) and 7 µl of DNA template in TTE buffer. The PCR reaction was performed for 3 min at 94°C, 35 cycles at 94°C for 30 s, 60°C for 30 s, and 70°C for 90 s, and 70°C for an additional 10 min. The 3 PCR products were mixed and purified using the Multiscreen-PCR Kit (Millipore), analyzed by gel electrophoresis, and quantified by OD measurement at 260 nm. The DNA was dried in a Speed Vac (Savant Instruments), and resuspended in spotting solution (0.1 M MES, 20% DMSO) to obtain a final concentration of 0.5 µg/µl. Preparation and hybridization of cDNA microarrays were performed according to Hot et al. (2003) with several modifications. PCR fragments were spotted three times onto 25×75 mm Codelink Activated Slides (Amersham) using the Affymetrix 417 Arrayer. Spotted slides were treated with blocking solution (50 mM Trizmahydrocloride, 50 mM Tris(hydroxymethyl)aminomethane, 0.3% ethanolamine, pH 9), containing 0.1% SDS at 50°C for 30 min. Slides were washed twice in distilled water for 2 min at room temperature, once in SSC 4× 0.1% SDS for 60 min at 50°C, once in distilled water for 1 min at room temperature, once in distilled water for 2 min at 95°C, and finally twice in distilled water for 2 min at room temperature, before being dried by centrifugation for 5 min at 180 g.