|
| Status |
Public on Jul 27, 2005 |
| Title |
dam mutS mock replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
E. coli dam mutS mutant strain GM5556
|
| Organism |
Escherichia coli |
| Characteristics |
As GM3819 dam-16::Kan but mutS215::Tn10
|
| Growth protocol |
Overnight cultures were diluted 1000-fold with fresh LB medium and cultured further. Log phase cultures were diluted to a cell density of 2 X 108 cells/ml in M9 salts and incubated at 37°C for two hours, after which they were resuspended in LB broth for 90 minutes.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Epicentre MasterPure RNA purification kit
|
| Label |
PEO-iodeacetyl-Biotin
|
| Label protocol |
Affymetrix protocol for E. coli sense arrays
|
| |
|
| Description |
Overnight culture was diluted 1000-fold with fresh LB medium and cultured further. Log phase culture was diluted to a cell density of 2 X 108 cells/ml in M9 salts and incubated at 37°C for two hours, after which it was resuspended in LB broth for 90 minutes. This treatment served as a mock treatment for cultures incubated with a chemical (cisplatin). Total RNA was isolated from cells by extraction using the MasterPure RNA Purification Kit (Epicentre Technologies) followed by DNAse digestion according to the manufacturer’s protocol. The isolated total RNA was quantitated by absorption at 260 nm (typical yield from a 15 ml culture was 250-500 µg of total RNA), and the purity was determined by the ratio of absorption values at 260/280nm. RNA quality was determined by formaldehyde agarose gel electrophoresis (1.2% agarose in FA Buffer pH 7.0 (20 mM 3-[N-morpholino]propanesulfonic acid, 5 mM sodium acetate, 1 mM ethylenadiaminetetraacetic acide (EDTA))) or by analysis on an Agilent 2100 Bioanalyzer. All samples visualized by gel electrophoresis or by the bioanalyzer electropherogram showed clear distinct bands correlating to 16S and 23S ribosomal RNA, indicating that no detectable RNA degradation occurred and that RNA integrity was maintained throughout the RNA isolation procedure. mRNA was enriched from total RNA as described in the Affymetrix GeneChip Expression Analysis Technical Manual for GeneChip E. coli Sense Genome Arrays.
|
| Data processing |
GCRMA normalization using Array Analyzer version 2.0.2 in S-Plus version 6.2 (Insightful)
|
| |
|
| Submission date |
Jul 25, 2005 |
| Last update date |
Oct 28, 2005 |
| Contact name |
Jennifer Robbins |
| E-mail(s) |
jrobbins@mit.edu
|
| Organization name |
Massachusetts Institute of Technology
|
| Department |
Biological Engineering
|
| Lab |
John Essigmann
|
| Street address |
77 Massachusetts Ave.
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL73 |
| Series (1) |
| GSE2999 |
Cisplatin-induced gene expression in DNA adenine methyltransferase (dam) and mismatch repair deficient E. coli |
|
