|
| Status |
Public on Jan 31, 2009 |
| Title |
S. aureofaciens two-component analysis |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Streptomyces coelicolor
|
| Organism |
Streptomyces coelicolor A3(2) |
| Characteristics |
Streptomyces coelicolor A3(2) (SCP1+) 104
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA from a stationary phase culture was purified by the salting out procedure (Pospiech, and Neumann, 1995) and had been sonicated to <2Kb.
|
| Label |
Cy3
|
| Label protocol |
Four to six micrograms of sonicated genomic DNA were used as template and this was denatured in the presence of 12 μl of 72% GC-content random hexamers in a total volume of 25μl at 100oC for ten minutes. The mixture was then snap-cooled on ice before adding the remaining reaction components: 1.5 μl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech), 4μl Klenow fragment (NEB #212), 5μl Klenow buffer, 0.5μl dNTP (4mM dATP, 4mM dTTP, 10mM dGTP, and 0.2mM dCTP), and 14μl ddH2O. The random primed labeling reaction was carried out for two to three hours at 37oC. Buffer exchange, purification and concentration of the DNA products was accomplished by three cycles of diluting the reaction mixture in 0.5ml TE buffer (10mM Tris and 1mM EDTA PH 8.0) and filtering though a Microcon-30 microconcentrators (Millipore).
|
| |
|
| Channel 2 |
| Source name |
Streptomyces aureofaciens
|
| Organism |
Kitasatospora aureofaciens |
| Characteristics |
Streptomyces aureofaciens ATCC10762
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA from a stationary phase culture was purified by the salting out procedure (Pospiech, and Neumann, 1995) and had been sonicated to <2Kb.
|
| Label |
Cy5
|
| Label protocol |
Four to six micrograms of sonicated genomic DNA were used as template and this was denatured in the presence of 12 μl of 72% GC-content random hexamers in a total volume of 25μl at 100oC for ten minutes. The mixture was then snap-cooled on ice before adding the remaining reaction components: 1.5 μl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech), 4μl Klenow fragment (NEB #212), 5μl Klenow buffer, 0.5μl dNTP (4mM dATP, 4mM dTTP, 10mM dGTP, and 0.2mM dCTP), and 14μl ddH2O. The random primed labeling reaction was carried out for two to three hours at 37oC. Buffer exchange, purification and concentration of the DNA products was accomplished by three cycles of diluting the reaction mixture in 0.5ml TE buffer (10mM Tris and 1mM EDTA PH 8.0) and filtering though a Microcon-30 microconcentrators (Millipore).
|
| |
|
| |
| Hybridization protocol |
The two DNA pools to be compared were mixed and applied to an array in a hybridization mixture that contained 3.68X SSC, 0.18% SDS, and 1μg yeast tRNA (total 16.3μl), which had been heated at 100oC for 5 min before being applied to array. Hybridization took place under a glass coverslip sealed by glue in a humidified Omnislide (Thermo Hybaid) at 60oC for twelve to fourteen hours.
|
| Scan protocol |
The slides were washed, dried and scanned for fluorescence using a GenePix TM 4000B scanner (Axon instruments).
|
| Description |
Kirby et al (2008) Antonie van Leewenhoek 94 173-186
|
| Data processing |
The average signal intensity and local background measurements were obtained for each spot on each array using GenePixPro software. The dataset was screened for aberrant spots and these were eliminated from the analysis after manual checking. Most genes are present in duplicate on the arrays and the signal from each pair of spots was inputted into the computer program available from ScanAlyze (Eisen et al., 1998; Gollub et al., 2003). The data was then processed into a mean log2 Cy3/Cy5 ratio format. The dataset was normalized for each array separately and outputted to Excel where after checking the alignment of the datasets from each array, a mean signal for each common gene was calculated. Based on Bentley et al., 2002, the mean signal and standard deviation for the core region of genes from SCO2050 to SCO5800 was calculated. The standard deviation was used to set a cut-off for gene absence at 2SD below the core mean. The ratio given are the normalized log2 ratio representing test organism/Streptomyces coelicolor ratio.
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| |
|
| Submission date |
Nov 16, 2008 |
| Last update date |
Dec 04, 2008 |
| Contact name |
Ralph Kirby |
| Organization name |
National Yang-Ming University
|
| Street address |
Li-Nong Road
|
| City |
Taipei |
| ZIP/Postal code |
112 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL7637 |
| Series (1) |
| GSE13621 |
Two-component signal transduction systems in Streptomyces and related organisms |
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