|
| Status |
Public on Apr 02, 2008 |
| Title |
LLNL virulence mechanism array version 2A LOD experiment: 3.1 pg S.aureus DNA |
| Sample type |
genomic |
| |
|
| Source name |
S.aureus genomic DNA
|
| Organism |
Staphylococcus aureus |
| Characteristics |
strain Mu50; GenBank accession NC_002758.2
|
| Biomaterial provider |
ATCC
|
| Growth protocol |
The bacterial culture pellets were grown according to the instructions from ATCC.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using the Epicentre DNA extraction kit according to the manufacturer's protocols.
|
| Label |
Cy3
|
| Label protocol |
DNA from one filter was used as a common background, to which varying quantities of fragmented S. aureus DNA were added. S. aureus DNA was quantified using a Quant-iT PicoGreen ds DNA kit (Invitrogen, Carlsbad, CA), serially diluted, and then spiked into 10 ng of aerosol sample DNA in quantities of 0.31 fg, 3.1 fg, 31 fg, 310 fg or 3.1 pg. We performed whole genome amplification of the combined samples (aerosol samples with spiked-in S. aureus DNA, plus one control pure aerosol sample) at 30oC for 16 hr using the REPLI-g whole genome amplification kit (Qiagen, Valencia, CA). The amplified material was inactivated at 65oC for 3 min and then purified using the QiaQuick PCR purification kit (Qiagen) to remove the primers and dNTPs. The entire amplified product was labeled with Cy3-random primer using the Klenow fragment.
|
| |
|
| Hybridization protocol |
For each hybridization, 4 μg of labeled DNA was mixed with Cy3- and Cy5-labeled CPK6 oligomers, NimbleGen hybridization components and hybridization buffer according to the manufacturer's protocols. The Arrays were hybridized with labeled DNA on a MAUI hybridization station (BioMicro Systems, Salt Lake City, UT) at 42 deg C for 16 hr. Arrays were washed with NimbleGen wash buffers I, II and III according to vendor protocols.
|
| Scan protocol |
Arrays were scanned using an Axon GenePix 4000B scanner at 5 μm resolution.
|
| Description |
NA
|
| Data processing |
Data were analyzed using custom software based on the R programming environment and BioConductor packages. Each probe was randomly spotted in three to five replicates to control for positional effects on the array. Data from replicate probes were summarized by the median of the log2-transformed intensities. No normalization or background correction was performed.
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| |
|
| Submission date |
Mar 31, 2008 |
| Last update date |
Apr 01, 2008 |
| Contact name |
Crystal Jaing |
| Organization name |
Lawrence Livermore National Laboratory
|
| Street address |
7000 East Ave.
|
| City |
Livermore |
| State/province |
CA |
| ZIP/Postal code |
94550 |
| Country |
USA |
| |
|
| Platform ID |
GPL6656 |
| Series (1) |
| GSE11010 |
Detection of virulence and antibiotic resistance gene families |
|
