|
| Status |
Public on Aug 23, 2007 |
| Title |
O157 no.6 (strain 990570) replicate 2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Genomic DNA from O157 strain 990570
|
| Organism |
Escherichia coli |
| Characteristics |
Enteroheamorragic E. coli
|
| Growth protocol |
Cells were grown to the stationary phase at 37ºC in Luria-Bertani medium.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was purified using the Genomic-tip 100/G and Genomic DNA buffer set (Qiagen) according to the manufacturer's instruction.
|
| Label |
Cy3
|
| Label protocol |
First, 3 µg of genomic DNA was labeled with aminoallyl-modified dUTP (Sigma) using the Bioprime DNA Labeling System (Invitrogen). DNAs were not sheared or digested by restriction enzymes prior to the labeling. The aminoallyl-labeled DNA was purified by Microcon YM-30 (Millipore), dried in a speed-vac, and resuspended in 10 µl of 50 mM NaHCO3. After adding 10 µl of dimethyl sulfoxide solution containing Cy3 or Cy5 monofunctional reactive dye (Amersham), the sample was incubated at room temperature in the dark for 1 h to allow the dye to couple with DNA. The fluorescently labeled DNA was finally purified by the Qiaquick PCR purification kit (Qiagen) into 30 µl elution buffer provided by the manufacturer.
|
| |
|
| Channel 2 |
| Source name |
O157 strain Sakai
|
| Organism |
Escherichia coli |
| Characteristics |
Enteroheamorragic E. coli
|
| Growth protocol |
Cells were grown to the stationary phase at 37ºC in Luria-Bertani medium.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was purified using the Genomic-tip 100/G and Genomic DNA buffer set (Qiagen) according to the manufacturer's instruction.
|
| Label |
Cy5
|
| Label protocol |
First, 3 µg of genomic DNA was labeled with aminoallyl-modified dUTP (Sigma) using the Bioprime DNA Labeling System (Invitrogen). DNAs were not sheared or digested by restriction enzymes prior to the labeling. The aminoallyl-labeled DNA was purified by Microcon YM-30 (Millipore), dried in a speed-vac, and resuspended in 10 µl of 50 mM NaHCO3. After adding 10 µl of dimethyl sulfoxide solution containing Cy3 or Cy5 monofunctional reactive dye (Amersham), the sample was incubated at room temperature in the dark for 1 h to allow the dye to couple with DNA. The fluorescently labeled DNA was finally purified by the Qiaquick PCR purification kit (Qiagen) into 30 µl elution buffer provided by the manufacturer.
|
| |
|
| |
| Hybridization protocol |
The Cy5-labeled and the Cy3-labeled DNA preparations (15 µl for each) were mixed with a 90-µl hybridization solution containing 6.4 x SSC, 0.64 % sodium dodecyl sulfate (SDS), and 1.3 mg/ml yeast tRNA. After incubation at 96ºC for 2 min, the denatured sample was applied to a microarray slide and incubated on an ArrayBooster hybridization apparatus (Advalytix AG, Brunnthal, Germany) at 50ºC for 16 h. The slide was then washed twice in 2x SSC/0.2% SDS at room temperature for 10 min, twice in 0.2x SSC/0.2% SDS at 50ºC for 5 min, and twice in 0.2x SSC at room temperature for 10 min. Finally, the slide was briefly rinsed with ethanol and dried by centrifugation.
|
| Scan protocol |
Scanned with a FLA-8000 scanner (Fuji Photofilm, Tokyo, Japan). The obtained data were analyzed by the ArrayVision 8.0 software (Imaging Research).
|
| Description |
none
|
| Data processing |
Spots with reference signal intensities lower than the local background (LBG) plus 5 standard deviations or with some spotting abnormalities were removed from analysis. Signal intensities of other spots were corrected by subtracting the LBG.
|
| |
|
| Submission date |
May 28, 2007 |
| Last update date |
Aug 23, 2007 |
| Contact name |
Yoshitoshi Ogura |
| E-mail(s) |
y-ogura@med.miyazaki-u.ac.jp
|
| Organization name |
University of Miyazaki
|
| Department |
Frontier science reserch center
|
| Street address |
Kihara 5200
|
| City |
Kiyotake |
| State/province |
Miyazaki |
| ZIP/Postal code |
889-1692 |
| Country |
Japan |
| |
|
| Platform ID |
GPL5329 |
| Series (1) |
| GSE7931 |
Genomic comparison in Enteroheamorragic E. coli O157 and non-O157 serotypes by comparative genomic hybridization. |
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