DNA sequencing was performed using a Big Dye fluorescent terminat

DNA sequencing was performed using a Big Dye fluorescent terminator and an ABI3770 capillary sequencer at the selleck chemical Plant Microbe Genomic Facility (The Ohio State University).

Microarray fabrication Details of the construction of the backbone version of the Salmonella array were described previously [13]. PCR products were purified using the MultiScreen PCR 96-well Filtration System (Millipore, Bedford, MA), and eluted in 30 μl of sterile water. Subsequently, the products were dried, resuspended in 15 μl 50% DMSO, and 5 μl were rearrayed into 384-well Epoxomicin datasheet plates for printing. Preparation of cDNA probes A 0.5 ml overnight culture of S. Typhimurium was used to inoculate 10 ml of LB and grown at 37°C, with shaking to an OD600 of 0.6–0.7. When inducing ectopically expressed preA (or vector

controls) with 10 mM arabinose, medium was buffered with 100 mM TrisHCl. Samples were transferred into chilled Falcon tubes containing 2 ml of 5% phenol/95% ethanol, incubated 15 min on ice, and cells were collected by centrifugation at 8000 g for 10 min at 4°C. Cells were lysed and RNA was collected, purified and DNase treated according to Promega SV Total RNA Isolation Kit (Promega, Madison, WI). RNA was checked for quantity and quality via gel electrophoresis or the Experion System (Bio-Rad, Hercules, CA). Cy3- and Cy5-dye-linked dUTP was directly incorporated during reverse transcription from total RNA to synthesize labeled cDNA

probes, based on the method described by [13] with the following modifications: 15–100 μg of total RNA and 2.4 μg of random hexamers were resuspended learn more in 30 μl of water, and subsequently the amounts and volumes of all components were doubled. Furthermore, 2 μl of RNase inhibitor (Invitrogen, Carlsbad, CA) was added to the reverse transcription, and the reaction incubated at 42°C for 2 h. After the first hour of incubation, 2 μl of additional Superscript II reverse transcriptase was added. Probes were purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA) and eluted in 50 μl sterile water. Subsequently, probes were dried down to 20 μl final volume. Hybridization and data acquisition Probes were mixed with equal volumes of 2× hybridization buffer containing 50% formamide, 10× SSC and 0.2% SDS, and boiled for 5 min. Probes were hybridized to Mirabegron the Salmonella array overnight at 42°C using a hybridization chamber (Corning, Corning, NY) submerged in water. Protocols suggested by the manufacturer for hybridizations in formamide buffer were applied for pre-hybridization, hybridization and post-hybridization wash processes. Scans were performed on an Affymetrix 428 Laser scanner (Affymetrix, Santa Clara, CA) using the Microarray suite 5.0 (Affymetrix) software. Data analysis The TIFF files where unstacked using ImageJ (NIH) and signal intensities were quantified using the QuantArray 3.

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