A 4X genomic library of P. syringae pv phaseolicola NPS3121 was constructed. 30% of the genomic library were randomly chosen and partially sequenced using a forward M13-primer in a single sequencing reaction. 2880 sequences with an average size of 531 pb were obtained. Using MUMmer system each sequence was aligned and annotated against the complete genome of P. syringae pv. phaseolicola 1448A (Joardar, 2005), this strategy allowed us to select 1911 clones that provided a 1X coverage of the genome, eliminate redundancy and to have information regarding the identity of each clone. The select clones were independently amplified by PCR using a M13 forward and reverse primers (5´-CCCAGTCACGACGTTGTAAAACGAC and 5´-AGCGGATAACAATTTCACACAGGAA, respectively). The PCR products were purified and transferred to 384 well source plates. The obtained DNA samples were adjusted at 120 ng/µl in 3X SSC and spotted onto amino Propyl Silane slides (Corning Inc., Corning, NY, U.S.A.) using the Virtek Chiprender Professional Arrayer. The array contained 1911 inserts with average size of 2.4 Kbp that cover at least 85% of the P. syringae pv phaseolicola NPS3121 genome. In the microarray were printed various positive and negative controls Lucidea Universal ScoreCard (Amersham) to monitor target labeling and hybridization efficiency, as well as many genes (argK, phtA, phtD, desI, phtL, phtMN, amtA) derived from our previous and ongoing research in phaseolotoxin synthesis. Additionally were printed PCR products for genes involved in the synthesis of type III secretion system (hrpRS, hrpTU, hrpOP, hrpJ, virPphA, avrPphC, avrPphD, avrPphE), quorum sensing (ahlI, ahlR, algD), global regulators (rpoD, gacA, rpoN, gacS, rsmA).
Support
glass
Coating
aminosilane
Description
All samples were printed in triplicate in a contiguous arrangement of 12 grids of 24 rows x 24 columns. The microarray was printed twice in the same slide. The printed conditions were: 20oC of temperature and 60% of humidity.
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