|
| Status |
Public on Aug 21, 2008 |
| Title |
E. coli 5 minute pH only dye swap treatment |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
E. coli, 5 minutes, no cadmium, pH 5
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
MG1655 strain of E. coli K-12
|
| Biomaterial provider |
University of Wisconsin Oshkosh
|
| Growth protocol |
E. coli was grown in M9 medium supplemented with 0.4% glucose. The cultures were grown on a rotary shaker (200 rpm) at 37 °C until the contents of the flask reached an OD600 of 0.3 (mid-log phase of growth). Each culture was divided into 25 mL aliquots, transferred to four 50 mL conical tubes, and centrifuged at 2540 x g for 12 minutes. The supernatant was decanted, and the cells were resuspended in 25 mL of M9 medium at pH 5. The cultures were incubated at 25 °C for 5 minutes with manual rotations of the flasks once per minute to resuspend the cells.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted and purified using a Masterpure RNA purification kit (Epicentre Technologies).
|
| Label |
Cy3
|
| Label protocol |
Total RNA was reverse transcribed with random primer mix and labeled with Cy 3 according to the 3DNA Array 900MPX kit manufacturer’s protocols (Genisphere).
|
| |
|
| Channel 2 |
| Source name |
E. coli, 5 minutes, no cadmium, pH 7
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
MG1655 strain of E. coli K-12
|
| Biomaterial provider |
University of Wisconsin Oshkosh
|
| Growth protocol |
E. coli was grown in M9 medium supplemented with 0.4% glucose. The cultures were grown on a rotary shaker (200 rpm) at 37 °C until the contents of the flask reached an OD600 of 0.3 (mid-log phase of growth). Each culture was divided into 25 mL aliquots, transferred to 50 mL conical tubes, and centrifuged at 2540 x g for 12 minutes. The supernatant was decanted, and the cells were resuspended in 25 mL of M9 medium at pH 7. The cultures were incubated at 25 °C for 5 minutes with manual rotations of the flasks once per minute to resuspend the cells.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted and purified using a Masterpure RNA purification kit (Epicentre Technologies).
|
| Label |
Cy5
|
| Label protocol |
Total RNA was reverse transcribed with random primer mix and labeled with Cy 5 according to the 3DNA Array 900MPX kit manufacturer’s protocols (Genisphere).
|
| |
|
| |
| Hybridization protocol |
The labeled cDNAs were hybridized together on a Corning Epoxide slide containing the Operon E. coli Genome Oligo Set Version 1.0 printed by the Microarray and Proteomics Facility, Department of Biological Sciences, University of Alberta. The hybridizations were performed in a hybridization chamber (Corning) in a dark, humidified chamber at 67 ºC for 13 hours. The slides were then washed in pre-warmed washing buffer 2X SSC containing 0.1% SDS for 20 minutes, 2X SSC for 20 minutes, and 0.2X SSC for 20 minutes.
|
| Scan protocol |
The arrays were scanned with a Versarray ChipReader (BioRad) with laser power at 75%, photomultiplier tube (PMT) sensitivity at 800 V, and detector gain at 1.
|
| Description |
Genes were represented in triplicate on each array.
|
| Data processing |
Spot intensities and locations were determined using TIGR Spotfinder, Version 3.1.1. All subsequent analyses were performed using the ma-anova package in the open-source statistical software package, R (www.r-project.org), Version 2.4.1. The data were normalized using the regional lowess method.
|
| |
|
| Submission date |
Aug 07, 2008 |
| Last update date |
Aug 21, 2008 |
| Contact name |
Craig Worden |
| Organization name |
UW Oshkosh
|
| Department |
Biology and Microbiology
|
| Street address |
800 Algoma Boulevard
|
| City |
Oshkosh |
| State/province |
WI |
| ZIP/Postal code |
54901 |
| Country |
USA |
| |
|
| Platform ID |
GPL6540 |
| Series (1) |
| GSE12373 |
Effect of pH on Cadmium Toxicity in Escherichia coli |
|
