|
| Status |
Public on Jun 01, 2006 |
| Title |
biological rep2 sample a IAA |
| Sample type |
RNA |
| |
|
| Source name |
Escherichia coli K12 growing in exponential growth phase
|
| Organism |
Escherichia coli K-12 |
| Characteristics |
Genotype: wild type strain MG1655
Age: 5 days old liquid colture, 4-times subcultivated
|
| Treatment protocol |
Exponentially growing E. coli K-12 (MG1655) cultures (OD600 = 0.6) was treated with an IAA solution to a final concentration of 0.5 mM. After two hours (OD600 = 1.2) different cell batches were aliquoted, frozen in liquid nitrogen for 5 min and stored at –80 °C for use in experiments.
|
| Growth protocol |
E.coli cells were grown aerobically at 37 °C in M9 minimal medium containing: 20 mg l-1 uracil, 1 mg l-1 thiamine, 0.4 % L-arabinose as carbon source, and supplemented with 40 mg l-1 casein acid hydrolysate.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified from cells grown in M9 arabinose medium using the RNeasy Mini-Kit from QIAGEN by following the manufacturer’s instructions.
|
| Label |
33P
|
| Label protocol |
33P-labelled probes were prepared using E. coli gene-specific primers (Sigma-Genosys) following the protocol provided by the manufacturer, except that the reaction mixture contained 40 mCi of [a-33P]dCTP (1,000 to 3,000 Ci mmol-1;Amersham Pharmacia), and 200 U of Superscript II (Promega) in 30 ml volume. Unincorporated-radiolabelled nucleotides were removed by applying the reaction mixture to a Sephadex G-25 gel filtration spin column.
|
| |
|
| Hybridization protocol |
After incubation for 2 h at 65 °C, the pre-hybridisation solution was replaced with 3 ml of hybridisation buffer containing the denatured radioactively labelled cDNA probes. Filter hybridisations were carried out for 16 h in a hybridisation oven. The filters were then washed three times, for 5 min each, at room temperature and then three times at 65 °C of warmed wash solution.
|
| Scan protocol |
The exposed phosphorimager screens were scanned at 100 mm resolution using a STORM 445 PhosphorImager (Molecular Dynamics).
|
| Description |
Gene expression data from late exponentially growing E. coli cells
|
| Data processing |
The signal intensities were measured by Image-Quant (version 3.0) software (Molecular Dynamics). Background values were measured in the surrounding region of the four corner grids containing E. coli genomic DNA, as a positive control, and empty spots. To avoid extreme intensity ratio for genes close to or below the detection limit, signal intensity values corresponding to a signal-to-background (S/B) ratio < 2.0 were scaled up to a value corresponding to the normalised background means.
|
| |
|
| Submission date |
May 30, 2006 |
| Last update date |
May 31, 2006 |
| Contact name |
Roberto Defez |
| E-mail(s) |
[email protected]
|
| Phone |
+390816132440
|
| Fax |
+390816132706
|
| URL |
http://www.igb.cnr.it
|
| Organization name |
National Research Council
|
| Department |
Institute of Genetics and Biophysics
|
| Lab |
Microbial Biotechnology
|
| Street address |
via Castellino 111
|
| City |
Napoli |
| ZIP/Postal code |
80131 |
| Country |
Italy |
| |
|
| Platform ID |
GPL189 |
| Series (1) |
| GSE4941 |
Expression data from untreated and IAA treated cells |
|
