|
| Status |
Public on Mar 31, 2008 |
| Title |
RIMD2210086 replicate2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
RIMD2210633
|
| Organism |
Vibrio parahaemolyticus |
| Characteristics |
sequenced strain
|
| Growth protocol |
Cells were grown to the stationary phase at 37ºC in Luria-Bertani containing medium3% NaCl .
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was purified using the DNeasy Tissue Kit (Qiagen) according to the manufacturer's instruction.
|
| Label |
Cy5
|
| Label protocol |
First, 2 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 phenol-chloroform extraction and ethanol precipitation. , Precipitated DNA was dried 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 CentriCep spin column (Princeton Sepatations Inc.).
|
| |
|
| Channel 2 |
| Source name |
RIMD2210086
|
| Organism |
Vibrio parahaemolyticus |
| Characteristics |
KP+, non-pandemic strain
|
| Growth protocol |
Cells were grown to the stationary phase at 37ºC in Luria-Bertani containing medium3% NaCl .
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was purified using the DNeasy Tissue Kit (Qiagen) according to the manufacturer's instruction.
|
| Label |
Cy3
|
| Label protocol |
First, 2 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 phenol-chloroform extraction and ethanol precipitation. , Precipitated DNA was dried 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 CentriCep spin column (Princeton Sepatations Inc.).
|
| |
|
| |
| Hybridization protocol |
Cy3- or Cy5- labeled probes (18 µl for each) were mixed, and 3 µl of 10% SDS, 6 µl of 25 mg/ml yeast tRNA and 15 µl of 20X SSC (3 M NaCl, 0.3 M trisodium citrate 2H2O, pH 7.0) were added to the mixture. After incubation at 96ºC for 2 min, the denatured sample was applied to a microarray slide and incubated on an MAUI hybridization chamber at 60ºC for 16 h. The slide was then washed twice in 2X SSC/0.1% SDS solution at 60°C for 10 min, twice in 0.2X SSC/0.1% SDS solution at room temperature for 10 min, and twice in 0.2X SSC solution at room temperature for another 10 min. Finally, the slide was briefly rinsed with ethanol and dried by centrifugation.
|
| Scan protocol |
Scanned with a ScanArrayExpress scanner (PerkinElmer). Obtained data were analyzed by the ScanArrayExpress software ver.3.0.
|
| Description |
RIMD2210086 replicate2
|
| Data processing |
LOWESS normalized, background subtracted data obtained from log2 of processed Cy3 signal/processed Cy5 signal.
|
| |
|
| Submission date |
Jan 04, 2008 |
| Last update date |
Jan 04, 2008 |
| Contact name |
Kaori Izutsu |
| Organization name |
Osaka University
|
| Department |
Research Institute for Microbial Diseases
|
| Lab |
Laboratory of Genomic Research on Pathogenic Bacteria
|
| Street address |
1-1 Yamadaoka
|
| City |
Suita |
| State/province |
Osaka |
| ZIP/Postal code |
565-0871 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6058 |
| Series (1) |
| GSE10020 |
Genomic comparison in Vibrio parahaemolyticus strains by comparative genomic hybridization. |
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