|
| Status |
Public on Jul 05, 2011 |
| Title |
tpx |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
TUV93-0
|
| Organism |
Escherichia coli O157:H7 |
| Characteristics |
strain: TUV93-0
genotype/variation: wild type
growth characteristics: mid exponential culture grown in MEM-HEPES supplemented with Fe and Glucose
|
| Growth protocol |
Escherichia coli O157 TUV93-0 was grown at 37 degrees C to OD 0.7 in MEM-HEPES supplemented with 0.1% Glucose and 250nM Fe(NO3)2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
An RNeasy minikit was used to prepare total RNA according to the manufacturer’s instructions (QIAGEN Ltd.). Any contaminating DNA was removed using a DNAase column kit (QIAGEN Ltd).
|
| Label |
Cy5
|
| Label protocol |
20 ug total RNA was converted to Cy3-labelled cDNA using the CyScribe Post-Labelling Kit (Amersham Biosciences/GE Healthcare) according to manufacturer’s instructions. Amino-allyllabelled nucleotides were incorporated into the cDNA by reverse transcription of the total RNA, followed by a direct chemical coupling of Cy3 NHS-dye esters to the amino-allyl-labelled cDNAs.
|
| |
|
| Channel 2 |
| Source name |
tpx
|
| Organism |
Escherichia coli O157:H7 |
| Characteristics |
strain: TUV93-0
genotype/variation: tpx mutant
growth chararceristics: mid exponential culture grown in MEM-HEPES supplemented with Fe and Glucose
|
| Growth protocol |
Escherichia coli O157 TUV93-0 was grown at 37 degrees C to OD 0.7 in MEM-HEPES supplemented with 0.1% Glucose and 250nM Fe(NO3)2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
An RNeasy minikit was used to prepare total RNA according to the manufacturer’s instructions (QIAGEN Ltd.). Any contaminating DNA was removed using a DNAase column kit (QIAGEN Ltd).
|
| Label |
Cy3
|
| Label protocol |
20 ug total RNA was converted to Cy3-labelled cDNA using the CyScribe Post-Labelling Kit (Amersham Biosciences/GE Healthcare) according to manufacturer’s instructions. Amino-allyllabelled nucleotides were incorporated into the cDNA by reverse transcription of the total RNA, followed by a direct chemical coupling of Cy3 NHS-dye esters to the amino-allyl-labelled cDNAs.
|
| |
|
| |
| Hybridization protocol |
Prior to hybridization, the slides were treated with the background-reducing Pronto! Pre-Soak System (Corning Life Sciences). The slides were incubated for 20 min in prewarmed Universal Pre-Soak solution at 42°C and washed twice in 0.1xSSC, 0.1% SDS for 30 s at room temperature. Slides were immediately transferred into prewarmed prehybridization solution (5xSSC, 0.1% SDS, and 0.1% bovine serum albumin) and incubated for 2–4 h at 42 °C. The microarray slides were finally washed at room temperature once in 0.1xSSC, 0.1% SDS for 1 min and twice in 0.1xSSC for 30 s, briefly dipped in water and then ethanol, and dried by centrifugation for 5 min at 800xg. For each experiment, equal quantities (80 pmol) of each Cy5- and Cy3-labeled cDNA were added to a final volume of 80 ul of hybridization solution containing 25% formamide, 10 mg of bovine serum albumin (fraction V) per ml, 5xSSC (1xSSC is 0.15 M NaCl, 0.015 M sodium citrate), 0.1% SDS, 8 ug of poly(A), and 1xDenhardt’s solution. The cDNA probes were denatured at 95 °C for 3 min and hybridized for 16 h at 42 °C. After hybridization was complete, the slides were washed in 2xSSC, 0.1% SDS at 42 °C for 2 min in 0.1xSSC, 0.1% SDS for 2 min at room temperature, and finally twice in 0.1xSSC for 2 min at room temperature. The microarray slides were dried by centrifugation for 5 min at 800g.
|
| Scan protocol |
Slides were scanned at 532 and 630 nm by using a Genepix 4000A scanner (Axon Instruments, Union City, CA).
|
| Description |
3 biological replicates
|
| Data processing |
Genespring 7.3.1 was used to obtain LOWESS normalised data that represents the average of the three replicate experiments.
Processed data for each replicate is attached as a supplementary txt file
|
| |
|
| Submission date |
Jul 19, 2010 |
| Last update date |
Jul 06, 2011 |
| Contact name |
Jai Justin Tree |
| E-mail(s) |
j.tree@unsw.edu.au
|
| Phone |
+61 2 938 59142
|
| Organization name |
University of New South Wales
|
| Department |
School of Biotechnology and Biomolecular Sciences
|
| Lab |
Tree lab
|
| Street address |
Rm s110 Bldg F25, UNSW, Gate 11 Botany St
|
| City |
Sydney |
| State/province |
NSW |
| ZIP/Postal code |
2033 |
| Country |
Australia |
| |
|
| Platform ID |
GPL3051 |
| Series (1) |
| GSE23001 |
Transciptional profile of Escherichia coli O157:H7 TUV93-0 inhibitor mutants in MEM-HEPES |
|
