|
| Status |
Public on Apr 01, 2007 |
| Title |
Hydrogen limitation vs. phosphate limitation slide 3000070253A rep 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA from Methanococcus maripaludis grown with 20 ml/min dihydrogen
|
| Organism |
Methanococcus maripaludis |
| Characteristics |
grown under hydrogen limitation
|
| Growth protocol |
cells were grown in chemostats in McCas media gassed with 20 ml/min dihydrogen
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen RNeasy
|
| Label |
Cy3
|
| Label protocol |
A variation of aminoallyl cDNA labeling (Ambion), 5-10 ug of total RNA was incubated with 4 ug of random decamers at 75 C for 7 min, reverse transcibed in the presence of 0.15mM aminoallyl UTP for 2 hours at 42 C,the RNA hydrolyzed by NaOH at 65 C for 15 min, neutralized with HEPES, percipitated and resuspended in coupling buffer, NHS-Cy3 was used to couple Cy3 to the aminoallyl UTP, and the reaction cleaned up with minelute columns
|
| |
|
| Channel 2 |
| Source name |
Total RNA from Methanococcus maripaludis grown under 15 mM phosphate
|
| Organism |
Methanococcus maripaludis |
| Characteristics |
grown under phosphate limitation
|
| Growth protocol |
cells were grown in chemostats in McCas media with 15 mM phosphate
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen RNeasy
|
| Label |
Cy5
|
| Label protocol |
A variation of aminoallyl cDNA labeling (Ambion), 5-10 ug of total RNA was incubated with 4 ug of random decamers at 75 C for 7 min, reverse transcibed in the presence of 0.15mM aminoallyl UTP for 2 hours at 42 C,the RNA hydrolyzed by NaOH at 65 C for 15 min, neutralized with HEPES, percipitated and resuspended in coupling buffer, NHS-Cy5 was used to couple Cy5 to the aminoallyl UTP, and the reaction cleaned up with minelute columns
|
| |
|
| |
| Hybridization protocol |
100-120 pmol of Cy3 and Cy5 labeled cDNA where mixed, dried, resuspended in hybridization buffer (Agilent), boiled 3 min, 30 sec on ice, placed on the slide, covered with a coverslip and incubated at 42 C for 14-16 hours in the dark. Slides where washed in 1xSSC + 0.2% SDS then 0.1xSSC + 0.2% SDS at 54 C, washed with 0.1xSSC at room temp then rinsed with water and dried under nitrogen gas.
|
| Scan protocol |
Scanned on a Molecular Dynamics Gen III array scanner
Images where quantified using TIGR spotfinder
|
| Description |
Biological replicate 1 of 4. Cultures grown under hydrogen limitation where compated to those grown under phosphate limitation
|
| Data processing |
Background subtraction followed by LOESS normalization using S+ array analyzer
|
| |
|
| Submission date |
Jan 15, 2007 |
| Last update date |
Jan 16, 2007 |
| Contact name |
John Leigh |
| Organization name |
University of Washington
|
| Department |
Microbiology
|
| Lab |
Leigh Lab
|
| Street address |
Box 357242
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
| |
|
| Platform ID |
GPL2509 |
| Series (1) |
| GSE6747 |
Methanococcus maripaludis response to hydrogen limitation |
|
