title: IntegratedGenomics_Ecoli_14K_v12-1
status: Public on Feb 19 2008
contact.city: Los Angeles
contact.first: Jason
contact.last: Ernst
contact.email: jason.ernst@ucla.edu
contact.state: CA
contact.address: 615 Charles E Young Dr South
contact.department: Biological Chemistry
contact.country: USA
contact.institute: UCLA
organism: Escherichia coli
manufacture_protocol: As described in (Tong et al. 2004, Biochemical and Biophysical Research Communications, 322: 347-354): "unique cDNA fragments corresponding to each predicted ORF of E. coli MG1655 were produced by PCR amplification from genomic DNA. To ensure that each primer pair would amplify the most unique 200–350 base pair region within each ORF, the PCR amplification product sequences were compared with all others in the genome by BLAST analysis. Sequences producing the minimum E values, relative to all other ORFs in E. coli, were selected. Of the 4485 E. coli ORFs listed in the ERGO database [Integrated Genomic, Chicago, IL], unique primers were found for 4442 ORFs (sequences available upon request). Most of the remaining ORFs represent genes that are shorter than 240 bp. Thus, each predicted ORF of E. coli MG1655 was represented by a fragment predicted to minimally cross-hybridize with other E. coli MG1655 sequences. These fragments varied in length from 200 to 350 bp, with a median length of ~300 bp. After two rounds of PCR amplification the final products were purified on 384-well format ArrayIt PCR purification Kits (TeleChem International). PCR products were resuspended in 15 μl spotting buffer (3× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 1.5 M betaine) and printed in triplicate onto amino-alkylsilane coated slides (Sigma) with an OmniGrid arrayer (Gene Machines) equipped with Telechem SMP3 split pins. The DNA was cross-linked to the slides with UV light and baked in a vacuum oven at 60 °C for at least two hours. Residual salt and unbound DNA were removed by rinsing the slides with 0.5% sodium dodecyl sulfate and water. The slides were stored desiccated at room temperature until use." Note that this array design is the same as IntegratedGenomics_Ecoli_14K_v1-12 except for a reordering of the location of the probes.