|
| Status |
Public on Apr 03, 2012 |
| Title |
BW25113 mqsR/pBS(Kan)-mqsR 2-1 in LB with ampicillin |
| Sample type |
RNA |
| |
|
| Source name |
BW25113 mqsR/pBS(Kan)-mqsR 2-1 in LB with ampicillin
|
| Organism |
Escherichia coli K-12 |
| Characteristics |
strains: K-12 BW25113
genotype/variation: mqsR/pBS(Kan)-mqsR 2-1
time: 1 h with 20 μg/mL ampicillin
medium: LB
cell type: Planktonic cells harvested at a turbidity of 0.5 at 600 nm, adjusted to the turbidity at 1, and then exposed to 20 μg/mL ampicillin for 1 h.
temp: 37ºC
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The overnight culture (0.25 ml) was used to inoculate 25 ml of fresh LB medium. Planktonic cells were grown to a turbidity of 0.5 at 600 nm in LB medium with 1 mM IPTG at 37 °C, adjusted the turbidity to 1, and exposed to 20 μg/mL ampicillin with 1 mM IPTG for 1 h. Cells were isolated by centrifuging at 0°C, and RNALater buffer (Ambion, Cat# AM7021) was added to stabilize RNA during the RNA preparation steps. After breaking the cells with a bead beater, the total RNA was isolated with Qiagen RNeasy mini Kit (Cat# 74104)
|
| Label |
biotin
|
| Label protocol |
Following affymetrix protocol.
cDNA was synthesized first using Promega M-MLV Reverse transcriptase (cat# M1705). After removing RNA, DNA fragmentation was performed to obtain and 50-200 base cDNA fragments. The fragmented cDNA was labelled with Biotin-ddUTP using Enzo BioArray Terminal Labeling Kit (Affymetrix, P/N 900181).
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| |
|
| Hybridization protocol |
Following affymetrix protocol.
Prepared hybridization cocktail for Single Probe Array (169 mini Format) with total 80 ul volume including 1X hybridization buffer, 50 pM B2 Control Oligo, 0.1 mg/mL Herring Sperm DNA, 0.5 mg/mL BSA, and at least 1 ug fragmented and labeled cDNA. After loading of hybridization cocktail in Affymetrix E. coli Genome 2.0 Array, the hybridization was performed at 45°C, with 60 rpm for 16 hours. After hybridization, the probe array was washed and stained using Affymetrix Genechip Fluidics Station 450 and the software GenomeChipOperating Software (GCOS).
|
| Scan protocol |
Following affymetrix protocol.
After washing and staining, the probe array was scanned using Affymetrix Genechip scanner GCS3000 7G system and the software GenomeChipOperating Software (GCOS).
|
| Data processing |
MAS 5.0 Expression Analysis Default Setting
|
| |
|
| Submission date |
Jul 29, 2011 |
| Last update date |
Apr 04, 2012 |
| Contact name |
Thomas K. Wood |
| E-mail(s) |
twood@engr.psu.edu
|
| Phone |
814-863-4811
|
| Organization name |
Pennsylvania State University
|
| Department |
Chemical Engineering & Biochemistry and Molecular Biology
|
| Lab |
Dr. Wood's Lab
|
| Street address |
Room 161 Fenske Laboratory, University Park
|
| City |
State College |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
| |
|
| Platform ID |
GPL3154 |
| Series (1) |
| GSE31054 |
Persistence of BW25113 mqsR producing MqsR 2-1 vs. producing wild-type MqsR with ampicillin |
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