|
| Status |
Public on Dec 30, 2011 |
| Title |
Wildtype Synechocystis sp. PCC 6803 under 24h high carbon, replica 1_2 |
| Sample type |
RNA |
| |
|
| Source name |
wildtype under 24h high carbon
|
| Organism |
Synechocystis sp. PCC 6803 |
| Characteristics |
strain: PCC 6803
genotype: wildtype
growth condition: high carbon
time: 24h
|
| Treatment protocol |
Physiological characterization of Synechocystis mutants was perfomed by axenic cultures of a defined optical density at 750 nm (OD750) of approx. 0.8, grown photoautotrophically in batch cultures ( 30 mm glass tubes with 5 mm glass tubes for aeration) at 29 °C under continuous illumination of 100 µmol photons m-2 s-1 (warm white light; Osram L58 W32/3) and bubbled with CO2-enriched air (5% CO2 in air, defined as high inorganic carbon- HC) in BG11 medium at pH 8.0, or bubbled with ambient air (0.035% CO2, defined as low inorganic carbon - LC) in BG medium ph 7.0. Growth was monitored by measurements of the OD750 (Ultraspec 3000 Pharmacia Biotech).
|
| Growth protocol |
Axenic cultures were grown on petri dishes at 30 °C under constant illumination of 30 µmol photons m-2 s-1 (warm white light; Osram L58 W32/3) using BG11 medium containing 0.8% agar (Rippka et al., 1079) buffered to pH 8.0 with 20 mM TES-KOH. Transformants were selected on media containing one of the following: 10 mg kanamycin mL-1 (Km) or 4 mg spectinomycin mL-1 (Sp). The further cultivation of mutants was done with one of the following: 50 mg Km mL-1, or 20 mg Sp mL-1.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells from 10 ml of culture were harvested by centrifugation at 4,000 rpm for 5 min at 4 °C and were immediately forzen at -80 °C . Total RNA was extracted after pre-treatment with hot phenol and chloroform using the High-Pure RNA isolation kit (Roche Diagnostics, Mannheim germany).
|
| Label |
Cy5
|
| Label protocol |
Direct cDNA labeling was done using fluorescent dyes Cy3 or Cy5 (Amersham, GE Healthcare, Munich, Germany).
|
| |
|
| Hybridization protocol |
Hybridization was perfomed according to the oligonucleotide protocol of Agilent. Aliquots of Cy3- or Cy5-labeled cDNA were denatured at 98°C for 3 min and mixed with 125 μL hybridization buffer and 25 μL control targets (Agilent).This hybridization solution was exposed to the microarray surface in Agilent hybridization chambers for 17 h in a rotating oven at 60°C. The slides were then washed according to the manufacturer's protocol.
|
| Scan protocol |
Arrays were scanned in an ozone-free room with a DNA microarray scanner G2565CA (Agilent Technologies), and feature extraction was performed with NimbleScan v2.5 (Roche NimbleGen).
|
| Description |
This Sample represents one channel of a dual-colour array.
MAD_251698910019_S01_GE2-v5_95_Feb07_1_2
|
| Data processing |
LOWESS REGIONAL normalized (R package,www.bioconductor.org), obtained from log2 procesed red signal/processed green signal.
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| |
|
| Submission date |
Aug 25, 2011 |
| Last update date |
Dec 30, 2011 |
| Contact name |
Floyd Wittink |
| E-mail(s) |
wittink@science.uva.nl
|
| Phone |
+31205257937
|
| Fax |
-
|
| URL |
http://www.micro-array.nl
|
| Organization name |
Universiteit van Amsterdam
|
| Department |
MAD
|
| Lab |
MAD
|
| Street address |
Kruislaan 318
|
| City |
Amsterdam |
| State/province |
- |
| ZIP/Postal code |
1098 SM |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL6546 |
| Series (1) |
| GSE31672 |
Comparison of carboxysomal (ccmM) and photorespiratory (glcD1/D2) mutants of Synechocystis sp. PCC 6803 using metabolic and transcriptomic phenotyping |
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