|
| Status |
Public on May 07, 2013 |
| Title |
250ng 16S rRNA gene amplicon of faecal sample used for the inoculation of the batch |
| Sample type |
other |
| |
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| Source name |
Stools
|
| Organism |
uncultured bacterium |
| Characteristics |
sample type: faecal sample
source: extracted from a faecal sample used for the inoculation of the batch
|
| Treatment protocol |
No treatment were studied in this experience
|
| Growth protocol |
Faecal samples and bioreactor samples were collected by the EA-CIDAM 4678, Faculté de Pharmacie, Université d'Auvergne, France.
|
| Extracted molecule |
other |
| Extraction protocol |
Total DNA was extracted from 1 human fecal sample and 4 bioreactor samples (Batch inoculum and 3 stage fermentor) using Qiagen’s DNA Stool Kit (Qiagen, West Sussex, UK). 10ng of sample was then used for PCR amplification. 16S rRNA genes were amplified using universal primers 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (TACGGYTACCTTGTTACGACT). PCR reactions were performed in a 50µl volume, using DreamTaq DNA polymerase (Fermentas, St. Leon-Rot, Germany). The PCR reaction consisted of an initial denaturation step at 95°C for 5min followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 40 s and elongation at 72°C for 2 min. A final extension step was performed at 72°C for 5 min. PCR product size was verified by electrophoresis with 1% (w/v) agarose gel and were purified using the MinElute PCR Purification Kit (Qiagen Ltd., UK) following manufacturer’s instructions and stored at -20°C.
|
| Label |
Cy3
|
| Label protocol |
For each sample, the purified 16S rRNA gene PCR products (1 µg) were labelled with either Cy3 or Cy5 using the Genomic DNA ULS labelling Kit (Agilent Technologies, Palo Alto, CA) following the manufacturer’s instructions.
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| |
|
| Hybridization protocol |
For microarray hybridization, 250ng of each labelled faecal sample were used. Hybridization was performed following the Agilent OligoaCGH hybridization protocol (Agilent Technologies, Palo Alto, CA) at 65°C for 24h.
|
| Scan protocol |
slides were scanned at a 3-µm resolution using a Surescan microarray scanner (Agilent Technologies, Palo Alto, CA).
|
| Description |
16S rRNA gene amplified from gDNA extracted from a faecal sample
|
| Data processing |
Pixel intensities were extracted using the “Feature Extraction” software (Agilent Technologies, Palo Alto, CA). No normalization step was performed and the retained intensity value for each probe was the ratio between the spot’s median intensity signals and the median of background signals.
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| |
|
| Submission date |
May 06, 2013 |
| Last update date |
May 07, 2013 |
| Contact name |
william tottey |
| E-mail(s) |
williamtottey@gmail.com
|
| Organization name |
EA-CIDAM 4678
|
| Street address |
28 place Henry Dunant
|
| City |
Clermont-Ferrand |
| ZIP/Postal code |
63000 |
| Country |
France |
| |
|
| Platform ID |
GPL16731 |
| Series (1) |
| GSE46677 |
Three-stage continuous culture system with a self-generated anaerobia to study the regionalized metabolism of the human gut microbiota |
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