Institute of Genome Research (IfG), Center of Biotechnology (CeBiTec), Bielefeld University, Germany
Manufacture protocol
Ps6kOLI1 oligonucleotide microarray represent 5.220 EST-clusters derived from 11930 Pisum sativum ESTs. These were donated by Hans Weber and Ruslana Radchuk (IPK Gatersleben; 8412 ESTs from cotyledons and seed coats), Aymeric Duclert (INRA Evry and Génoplante; 2816 ESTs from stipules, epicotyls, leaves, stems and seeds) and Julie Hofer (JIC Norwich; 702 ESTs from lateral and apical shoot tips before and during flowering). Automatic blast comparisons against GenBank "nr", the TIGR M. truncatula and Glycine max Gene Indices as well as the directionally cloned JIC and IPK collections were used to establish the orientation of the EST clusters obtained and all clusters were subsequently converted to "sense" orientation. In case of 300 clusters with ambiguous directionality due to (1) lack of polyA tails or (2) lack of significant homologies, both "sense" and "antisense" orientations were selected. Including a collection of 26 marker genes for P. sativum seed, stem and flower development (supplied by J. Hofer, JIC Norwich; C. Rameau, INRA Versailles and R. Radchuk, IPK Gatersleben) a total of 4946 non-redundant EST clusters are represented on the arrays. All oligonucleotides were spotted in 3 replicates. Oligonucleotides serving as controls were spotted in more replicates in some cases. Control oligos detecting different transgenes (bar, nptII, gusA, gfp, luc, hph) • PSGAPDH-5-100/-C-100/-3-100 oligonucleotides covering the 5', the central and the 3' region of the GAPDH gene that can serve as controls for the processivity of labeling • PSGAPDH-C-100 to C-74 oligonucleotides that can serve as controls to assess crosshybridization. • NCPS01- NCPS06 oligonucleotides serve as alien controls to detect unspecific hybridization. • Alien1- Alien10 oligonucleotides serve as alien controls to detect unspecific hybridization • PCPS01 – PCPS26 oligonucleotides serve as positive controls representing expressed genes • EMPTYWELLS only containing spotting buffer. These can be used to adjust the flagging for lowintensity ("empty") spots that give signals very close to background More information : www.cebitec.uni-bielefeld.de/legume/microarrays/microarrays.html
Support
glass
Coating
aminosilane
Description
Each microarray contains 5.376 triplicate spots (total number of spots: 16.128) in 48 grids of 19 rows and 18 columns; all triplicates are spotted in the same grid. The 48 grids were arrayed in a 4x12 pattern of 4 metacolumns and 12 metarows. In each grid, 6 positions are not arrayed leading to 288 positions that do not contain spots across the arrays. The spots contain 5276 different 70mer oligonucleotides. A total of 42 empty wells containing no 70mers but only spotting buffer are distributed across the Ps6kOLI1 arrays in 3 duplicates giving a total of 126 background controls designated EMPTYWELLS.
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