|
| Status |
Public on Apr 26, 2007 |
| Title |
EHEC LB 7 h biofilm cells |
| Sample type |
RNA |
| |
|
| Source name |
7 h biofilm cells
|
| Organism |
Escherichia coli |
| Characteristics |
RNA extracted from biofilm cells of EHEC wild type after 7h of growth in LB and 0.1 % DMF with glass wool
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To lyse the cells, 1.0 mL RLT buffer (Qiagen, Inc., Valencia, CA) and 0.2 mL 0.1 mm zirconia/silica beads (Biospec) were added to the frozen bead beater tubes containing the cell pellets. The tubes were closed tightly and beat for 50 seconds at the maximum speed in a mini bead beater (cat. no. 3110BX, Biospec). The total RNA was isolated by following the protocol of the RNeasy Mini Kit (Qiagen) including an on-column DNase digestion with RNase-free DNase I (Qiagen).
|
| Label |
biotin
|
| Label protocol |
The total RNA samples were first converted into cDNA through a reverse transcription reaction with poly-A RNA controls spiked into the same reaction mixture to monitor the entire target labeling process. The cDNA was then digested with DNase I (Amersham Biosciences) to produce 50-200 bp fragments, which was checked by running the fragmented cDNA on a 2% agarose gel. The fragmented cDNA was labeled at the 3’ termini by the Enzo BioArray Terminal Labeling Kit with Biotin-ddUTP (Affymetrix, P/N 900181).
|
| |
|
| Hybridization protocol |
The biotin-labeled target was hybridized to the Affymetrix GeneChip E. coli antisense array at 45°C for 16 hour at 60 rpm using the Hybridization Oven 640 (Affymetrix), then a three-step fluorescent staining was conducted using the Fluidics Station 450 (Affymetrix) during the washing and staining procedure.
|
| Scan protocol |
The microarray was scanned at 570 nm to get an image file by the GeneChip Scanner 3000 (Affymetrix). Using GeneChip® Operating Software, total cell intensity was scaled automatically in the software to an average value of 500.
|
| Description |
RNA extracted from biofilm cells of EHEC wild type after 7 h of growth in LB and 0.1 % DMF with glass wool
|
| Data processing |
MAS 5.0 Expression Analysis Default Setting
|
| |
|
| Submission date |
Oct 30, 2006 |
| Last update date |
Apr 26, 2007 |
| Contact name |
Jintae Lee |
| E-mail(s) |
[email protected]
|
| Phone |
1-979-845-1744
|
| Fax |
1-979-845-6446
|
| Organization name |
Texas A&M University
|
| Department |
Chemical Engineering
|
| Street address |
MS 3122
|
| City |
College Station |
| State/province |
TX |
| ZIP/Postal code |
77843-3122 |
| Country |
USA |
| |
|
| Platform ID |
GPL3154 |
| Series (1) |
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