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Status |
Public on Apr 01, 2007 |
Title |
Rapid speed under phosphate limitation vs. slow speed under phosphate limitation slide 3000070023B rep 1 |
Sample type |
RNA |
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|
Channel 1 |
Source name |
Total RNA from Methanococcus maripaludis grown with 0.15 mM phosphate at 0.19 per hour dilution rate
|
Organism |
Methanococcus maripaludis |
Characteristics |
grown at rapid speed under phosphate limitation
|
Growth protocol |
cells were grown in chemostats in McCas media with 0.15 mM phosphate at 0.19 per hour dilution rate
|
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
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Channel 2 |
Source name |
Total RNA from Methanococcus maripaludis grown with 0.15 mM phosphate at 0.042 per hour dilution rate
|
Organism |
Methanococcus maripaludis |
Characteristics |
grown at slow speed under phosphate limitation
|
Growth protocol |
cells were grown in chemostats in McCas media with 0.15 mM phosphate at 0.042 per hour dilution rate
|
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
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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 Perkin-Elmer Scan-Array Express Images where quantified using TIGR spotfinder
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Description |
Biological replicate 1 of 3. Cultures grown at rapid speed where compared to those under slow growth, both under phosphate limitation
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Data processing |
Background subtraction followed by LOESS normalization using S+ array analyzer
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Submission date |
Jan 15, 2007 |
Last update date |
Jan 16, 2007 |
Contact name |
John Leigh |
Organization name |
University of Washington
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Department |
Microbiology
|
Lab |
Leigh Lab
|
Street address |
Box 357242
|
City |
Seattle |
State/province |
WA |
ZIP/Postal code |
98195 |
Country |
USA |
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|
Platform ID |
GPL2509 |
Series (1) |
GSE6747 |
Methanococcus maripaludis response to hydrogen limitation |
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