|
| Status |
Public on May 23, 2009 |
| Title |
tpbA mutant (rep2) LB biofilm 7 h 37C |
| Sample type |
RNA |
| |
|
| Source name |
tpbA mutant (rep2) LB biofilm 7 h 37C
|
| Organism |
Pseudomonas aeruginosa |
| Characteristics |
genotype: tpbA (PA14_13660) mutant
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The overnight culture (2.5 ml) was used to inoculate 250 ml of fresh LB medium with 10 g of submerged glass wool (Corning Glass Works, Corning, NY) for forming biofilm. After incubation for 15 h at 37°C with shaking (250 rpm), the glass wool was carefully and quickly removed and rinsed with 100 ml of sterile 0.85% NaCl solution at 0°C. Biofilm cells were removed by sonicating the glass wool in 200 ml of sterile 0.85% NaCl solution at 0°C. After breaking the cells with a bead beater, and the total RNA was isolated with Qiagen RNeasy mini Kit (Cat# 74104).
|
| Label |
biotin
|
| Label protocol |
Following affymetrix protocol.
cDNA was synthesized first using Promega M-MLV Reverse transcriptase (cat# M1705). After removing RNA, DNA fragmentation was performed to obtain and 50-200 base cDNA fragments. The fragmented cDNA was labelled with Biotin-ddUTP using Enzo BioArray Terminal Labeling Kit (Affymetrix, P/N 900181).
|
| |
|
| Hybridization protocol |
Following affymetrix protocol.
Prepared hybridization cocktail for Single Probe Array (49 Format) with total 200 ul volume including 1X hybrization buffer, 50 pM B2 Control Oligo, 0.1 mg/mL Herring Sperm DNA, 0.5 mg/mL BSA, and at least 1 ug fragmented and labelled cDNA.
After loading of hybridization cocktail in Affymetrix E. coli Antisense Genome Array, the hybridization was performed at 45ºC, with 60 rpm for 16 hours.
After hybridization, the probe array was washed and stained using Affymetrix Genechip Fluidics Station 450 and the software GenomeChipOperating Software (GCOS).
|
| Scan protocol |
Following affymetrix protocol.
After washing and staining, the probe array was scanned using Affymetrix Genechip scanner GCS3000 7G system and the software GenomeChipOperating Software (GCOS).
|
| Description |
RNA extracted from biofilm cells of PA14_tpbA mutant after 7 hours of growth at 37C in LB with glasswool
|
| Data processing |
MAS 5.0 Expression Analysis Default Setting
|
| |
|
| Submission date |
Dec 08, 2008 |
| Last update date |
May 23, 2009 |
| Contact name |
Akihiro Ueda |
| E-mail(s) |
[email protected]
|
| Phone |
979-845-1744
|
| Fax |
979-845-6446
|
| Organization name |
Texas A&M University
|
| Department |
Chemical Engineering
|
| Street address |
3122 TAMU
|
| City |
College Station |
| State/province |
TX |
| ZIP/Postal code |
77843-3122 |
| Country |
USA |
| |
|
| Platform ID |
GPL84 |
| Series (1) |
| GSE13871 |
Pseudomonas aeruginosa PA14 wild-type vs the tpbA (PA14_13660) mutant |
|
