|
| Status |
Public on Mar 01, 2013 |
| Title |
100ng 16S rRNA gene amplicon of a mock community |
| Sample type |
other |
| |
|
| Source name |
Mock community
|
| Organisms |
Escherichia coli; Bacteroides fragilis; Blautia coccoides; [Clostridium] leptum; Lactobacillus acidophilus |
| Characteristics |
sample type: mock community [artificial bacterial DNA mix]
|
| Treatment protocol |
No treatment were studied in this experience
|
| Growth protocol |
Faecal samples were collected by the Department of Microbiology and Alimentary Pharmabiotic Centre (Cork, Ireland) from three elderly patients (176, 204 and 205). The mock community was performed after culture of each individual bacterial strain in their reference medium at their optimal temperature for several days.
|
| Extracted molecule |
other |
| Extraction protocol |
Total DNA was extracted from three human faecal samples using Qiagen’s DNA Stool Kit (Qiagen, West Sussex, UK). Total genomic DNA was extracted from pure bacterial cultures using DNeasy Blood and Tissue 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 |
Cy5
|
| Label protocol |
For each sample (faecal samples and the mock community), 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, 100 ng of labelled artificial bacterial DNA mix and 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 bacterial strains.
Sample name: Mock community
|
| 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 |
Feb 28, 2013 |
| Last update date |
Mar 01, 2013 |
| Contact name |
Nicolas Parisot |
| Organization name |
EA 4678 CIDAM
|
| Street address |
28 place Henri Dunant
|
| City |
Clermont-Ferrand |
| ZIP/Postal code |
63001 |
| Country |
France |
| |
|
| Platform ID |
GPL16731 |
| Series (1) |
| GSE44752 |
The human Gut Chip “HuGChip”, an explorative phylogenetic microarray for determining gut microbiome diversity at Family level |
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