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Status |
Public on Apr 03, 2012 |
Title |
BW25113 wild-type stationary phase OD 3.0 |
Sample type |
RNA |
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Source name |
Channel 1
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Organism |
Escherichia coli |
Characteristics |
strain: E. coli BW25113 wild-type growth phase: stationary phase OD 3.0 time: 0h sample type: starting culture medium: LB cell type: Planktonic cells inoculated at OD 0.05 at 600nm, grown to OD 3.0 at 600nm, exposed to 5 ug/mL ampicillin for 30 min. temp: 37C
|
Extracted molecule |
total RNA |
Extraction protocol |
One-day old single colony was inoculated in 25 mL of LB for 16 h at 37C at 250 rpm. Overnight culture was diluted to be OD 0.05 at 600nm in 25 mL in LB and incubated at 37C at 250 rpm until OD 3.0 at 600nm (harvested at this OD). Ampicillin 5 µg/mL was then added directly to 22 mL of culture and incubated for an additional 30 mins. Cell pellet was collected by centrifuging at 8500 rpm at 4C for 2 min. Supernatant was discarded and the cell pellet was resuspended with 1 mL RNAlater. Then the sample was centrifuged and the supernatant was discarded, the final cell pellet was immediately frozen in dry ice and stored at -80C. After breaking the cells with a bead beater, and 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).
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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).
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Description |
RNA extracted from stationary phase cells of E. coli BW25113 wild-type exposed to ampicillin 5 ug/mL for 30 mins
|
Data processing |
MAS 5.0 Expression Analysis Default Setting
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Submission date |
Nov 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 (2) |
GSE34028 |
BW25113 H202 and delta rpoS persister cells vs. stationary phase BW25113 wild-type |
GSE34177 |
BW25113 Rif pre-treated persister cells vs. stationary phase BW25113 wild-type |
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