|
| Status |
Public on Jul 20, 2010 |
| Title |
BW25113 (rpoS-) log ethanol shock, biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Escherichia coli BW25113（rpoS-）
|
| Organism |
Escherichia coli |
| Characteristics |
genotype: rpoS-
genetic background: BW25113
|
| Treatment protocol |
E.coli cells grown in M9 media with at 37º C until stationary phase (2.0), then were treated with 15%（v/v） ethanol shock for 15 min after that the cells were harvested to extract RNA.
|
| Growth protocol |
E.coli cells grown with aeration in M9 media at 37º C until stationary phase (2.0) and were treated with 15%（v/v） ethanol shock for 15 min , after that the cells were harvested to extract RNA. M9 medium recipe is as follow: NH4Cl 0.5g Na2HPO4 3.0g KH2PO4 1.5g 2% (w/v)Dextrose per 1 liter.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the commercial product TRIzol Reagent (Invitrogen, Carlsbad, CA).Disrupt about 107cells with a homogenizer with 1ml TRIzol Reafent on ice. Store the homogenate for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes. Next, supplement the homogenate with 0.2 ml chloroform per 1 ml of TRI Reagent, cover the samples tightly and shake vigorously for 15 seconds. Store the resulting mixture at room temperature for 2-15 minutes and centrifuge at 12,000 g for 15 minutes at 4 C. Following centrifugation, the mixture separates into a lower red phenol-chloroform phase, interphase and the colorless upper aqueous phase. Transfer the aqueous phase to a fresh tube. Precipitate RNA from the aqueous phase by mixing with isopropanol. Use 0.5 ml of isopropanol per 1 ml of TRI Reagent used for the initial homogenization. Store samples at room temperature for 5-10 minutes and centrifuge at 12,000 g for 8 minutes at 4 - 25 C. Remove the supernatant and wash the RNA pellet ( by vortexing) with 75% ethanol and subsequent centrifugation at 7,500 g for 5 minutes at 4 - 25 C. Add at least 1 ml of 75% ethanol per 1 ml TRI Reagent used for the initial homogenization. Remove the ethanol wash and briefly air-dry the RNA pellet for 3 - 5 min. Dissolve RNA in DEPC-treated water by passing solution a few times through a pipette tip. Using an on-column DNase digestion with RNase-free DNase I (Qiagen) to purify the total RNA.
|
| Label |
biotin
|
| Label protocol |
Affymetrix Technical Manual, Prokaryotic Sample and Array Processing
|
| |
|
| Hybridization protocol |
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Drosophila Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
The data were collected by using Affymetrix GeneChip Scanner 3000.
|
| Description |
n/a
|
| Data processing |
Affymetrix GeneChip Operating Software (GCOS) Version 1.4
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| |
|
| Submission date |
Aug 05, 2009 |
| Last update date |
Aug 12, 2009 |
| Contact name |
Ming Chen |
| E-mail(s) |
chenmingbio@hotmail.com
|
| Organization name |
Biotechnology Research Institute, Chinese academy of Agricultural Sciences
|
| Street address |
Zhongguancun Nandajie 12th,Haidian Zone
|
| City |
Beijing |
| ZIP/Postal code |
100081 |
| Country |
China |
| |
|
| Platform ID |
GPL3154 |
| Series (1) |
| GSE17526 |
Transcriptional responses of Escherichia coli rpoS- BW25113 vs. wild-type BW25113 under 15% ethanol shock in log phase |
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