|
| Status |
Public on Sep 15, 2015 |
| Title |
1221-U101 replicate 3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Lab Stock
|
| Organism |
Campylobacter jejuni RM1221 |
| Characteristics |
sample type: control (representing 5 independent DNA preps of C. jejuni RM1221)
|
| Growth protocol |
The control and the sample strains (sample strains first isolated from pig) were grown in blood agar plates {Blood Agar Base no: 2 (CM271, Oxoid, UK) supplemented with 5% defibrinated horse blood (TCS, UK)} overnight at 42ºC in an anaerobic jar (Oxoid, UK) vacuumed to - 22 psi and atmospheric pressure was restored with a gas mixture consisting of 85 % v / v nitrogen, 10 % v / v carbon dioxide and 5 % v / v hydrogen (Air Products, Crewe, UK).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated using Qiagen genomic extraction kit (using genomic DNA buffer set Cat no: 19060 and Genomic tip20/G Cat No: 10223) according to the manufacturer’s instructions.
|
| Label |
Cy5
|
| Label protocol |
1. Set up a labelling reaction by mixing. 9 µg Random primers (Promega, UK). 3 µg Template genome. Make the volume to 41.5 µl by adding nuclease free water. 2. Heat at 95° C for 5 min and snap cool. 3. Add final reaction elements. 41.5 µl heated template. 1.5 µl dye-dCTP 1 µl dNTP mix (5mM dA, dT, dG, 2mM dC) 5 µl Klenow Reaction mix. (Fermentas, UK) 1 µl Klenow @ 10 U/ µl (Fermentas, UK) 4. Heat at 37° C for 1.5 hrs. 5. Min elute reaction clean up kit was used to clean up the reaction mix according to the manufacturers instructions (Qiagen, UK).
|
| |
|
| Channel 2 |
| Source name |
Campylobacter cells isolated from pig intestinal cells
|
| Organism |
Campylobacter jejuni |
| Characteristics |
strain: U101
sample type: test
|
| Growth protocol |
The control and the sample strains (sample strains first isolated from pig) were grown in blood agar plates {Blood Agar Base no: 2 (CM271, Oxoid, UK) supplemented with 5% defibrinated horse blood (TCS, UK)} overnight at 42ºC in an anaerobic jar (Oxoid, UK) vacuumed to - 22 psi and atmospheric pressure was restored with a gas mixture consisting of 85 % v / v nitrogen, 10 % v / v carbon dioxide and 5 % v / v hydrogen (Air Products, Crewe, UK).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated using Qiagen genomic extraction kit (using genomic DNA buffer set Cat no: 19060 and Genomic tip20/G Cat No: 10223) according to the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
1. Set up a labelling reaction by mixing. 9 µg Random primers (Promega, UK). 3 µg Template genome. Make the volume to 41.5 µl by adding nuclease free water. 2. Heat at 95° C for 5 min and snap cool. 3. Add final reaction elements. 41.5 µl heated template. 1.5 µl dye-dCTP 1 µl dNTP mix (5mM dA, dT, dG, 2mM dC) 5 µl Klenow Reaction mix. (Fermentas, UK) 1 µl Klenow @ 10 U/ µl (Fermentas, UK) 4. Heat at 37° C for 1.5 hrs. 5. Min elute reaction clean up kit was used to clean up the reaction mix according to the manufacturers instructions (Qiagen, UK).
|
| |
|
| |
| Hybridization protocol |
The slides used for the genome analysis were γ APS coated, A+ bar-coded Schott nexterion slides. The slides were printed according to campy dense array gal by Microgrid II 600 robot using TAS software version V2.4.03. They had 12 pins with a 26 x 26 pin grid configuration. The spacing between the spots was 0.165mm and the slides were prepared at 60% humidity. Each slide was printed with duplicate sets of oligos for NCTC 11168, RM1221 and RM2228. A sample of each labelled probe (one cy3, one cy5) for each slide was taken to be hybridized which contained 500ng of labelled DNA and placed them together in a 0.5 ml tube. The volume was adjusted to 4.5µl total volume by adding nuclease free water. To each tube 0.5 µl of Fragmentation Buffer was added before being incubated at 70OC (in Thermo Cycler) for 2 min followed by 0.5 µl of Stop Solution (Ambion Kit 8740). This was followed by the addition of 2µl Lithium Heperin (to reduce non specific hybridization). Add 100 µl of Schott 1x Hybridization Buffer which was pre-warmed to 50OC and added to the probe carefully to avoid introducing any air bubbles. The total volume was 107.5 µl. The DNA microarray slides were blocked before hybridisation by using blocking solution (5xSSC, 0.2%SDS, 1%BSA). After blocking, the slides were washed in ultra pure water and then in 100% ethanol. The prepared slides were kept in the slide holder and were inserted into the TECAN HS 4800 chamber. Approximately 5 min before the probes were inserted they were heated to 95o C for 2 min and then spun at 15.000rpm for 5 minutes in a microcentrifuge to pellet up any impurities. The probe was injected into the hybridisation chamber. The hybridisation was done at 45º C for 16h with gentle agitation. After hybridisation the slides were washed in 1 X SSC buffer with 0.6% SDS for 2 min, followed by two washes in 0.06 X SSC buffer, each for 2 min. The slides are then dried at 23º C with nitrogen.
|
| Scan protocol |
Slides were scanned using Axon Genepix 4200AL scanner (Molecular Devices Corporation, Sunnyvale, CA ) at 532 (Cy3) and 635 (Cy5) excitation wavelengths with 5 µm resolution. The slides were scanned using the Auto PMT function with the threshold for saturation set at 0.05%. Fluorescent spot intensities were quantified using Genepix Pro 6.0 software. Spots were excluded from further analysis if they contain anomalous spot morphology or were within regions of non-specific fluorescence.
|
| Description |
Biological replicate 3 of 3. Control 1221 to U101
|
| Data processing |
The data was analysed by Genespring GX 7.3 and the oligos which have more than 2 fold change and p<0.05 were selected for further studies.
|
| |
|
| Submission date |
Aug 18, 2009 |
| Last update date |
Sep 15, 2015 |
| Contact name |
Amy John |
| E-mail(s) |
asj918@gmail.com
|
| Organization name |
University of Nottingham
|
| Street address |
Loughborough
|
| City |
Leicestershire |
| ZIP/Postal code |
LE12 5RD |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL9022 |
| Series (1) |
| GSE17701 |
Genome analysis of C. jejuni and C. coli isolated from different regions of the same pig |
|
