|
| Status |
Public on Nov 01, 2006 |
| Title |
P4XB2 Mutant (1993) in Arginine |
| Sample type |
RNA |
| |
|
| Source name |
E.Coli, strain P4XB2 mutant, grown in presence of arginine(100 ug/ml)
|
| Organism |
Escherichia coli |
| Characteristics |
E.Coli, strain P4XB2 mutant, grown in presence of arginine(100 ug/ml)
|
| Biomaterial provider |
Marina Caldara VUB Pleinlaan 2, 1050 Brussels,Belgium.
|
| Growth protocol |
Cells used for expression analysis were grown in minimal medium (Glansdorff, 1965) supplemented with 0.5% glucose (w/v), L-methionine (100ug ml-1) and with L-arginine (100ug ml-1). For expression analysis, cells were grown in a rotary shaker at 37°C and harvested by centrifugation at mid-log phase ( (OD) 660nm = 0.5) and the metabolism was quenched in liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using a RNeasy® RNA isolation kit, according to the manufacturer’s specifications (QIAGEN, Germany) and stored in RNase-free water at –80°C. RNA concentration was determined by UV-spectrometry and its quality controlled by agarose gel electrophoresis.
|
| Label |
Biotin
|
| Label protocol |
Total RNA was controlled for its integrity and purity using a Bioanalyzer 2100 (Agilent) and a Nanodrop spectrophotometer. Total RNA (10µg) showing no signs of degradation or impurities, was supplemented with Poly-A RNA controls and reverse transcribed to cDNA using random primers. After RNA degradation with NaOH, cDNA was purified (MinElute PCR Purification Kit®, QIAGEN), analyzed for yield (30-120µg) and purity (260nm/280nm and 260nm/230nm absorption ratios >1.8), then fragmented using DNase-I (Amersham Pharmacia Biosciences) and labelled terminally with biotinylated GeneChipÒ DNA Labeling Reagent (Affymetrix, UK).
|
| |
|
| Hybridization protocol |
A minimum of 1mg fragmented probe was resuspended in 80 ml hybridization buffer containing 3nM of control oligo B2 (Affymetrix, UK). Hybridization was performed in a rotisserie oven at 45°C for 16 hours. The chips were washed and stained in a GeneChip® Fluidics Station 400™ (Affymetrix, UK) using the Mini_prok2v1 protocol
|
| Scan protocol |
The chips were scanned with the GeneChip® Scanner 3000 (Affymetrix, UK).
|
| Description |
no additional information
|
| Data processing |
Initial data analysis was performed using the GeneChip® Operating Software. Microarray quality control parameters were as follow: noise (RawQ) less than 5, background signal less than 40 (100 target intensity for array scaling), consistent numbers of genes detected as present across arrays.
|
| |
|
| Submission date |
Apr 26, 2006 |
| Last update date |
Sep 06, 2006 |
| Contact name |
Rekin's Janky |
| E-mail(s) |
[email protected]
|
| Organization name |
VIB
|
| Department |
Nucleomics Core
|
| Street address |
Herestraat 49 Box 816
|
| City |
Leuven |
| ZIP/Postal code |
B-3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL3154 |
| Series (1) |
| GSE4724 |
Transcriptome analysis of the arginine regulon in E.coli |
|
