year: 2007 sample type: Pristine sample name: 32D source: Pristine active layer soil from a ditch nearby Eureka weather station
Extracted molecule
genomic DNA
Extraction protocol
Soil DNA was extracted from a 10 g soil sub-sample using the MoBio PowerMax Soil DNA Isolation kit (MoBio Laboratories, Carlsbad, CA).
Label
Cy5
Label protocol
Total DNA extracted from all soil samples was submitted to a PCR procedure with eubacterial 16S rRNA gene primers F1-R13, with an annealing temperature of 50°C. RT-PCR products were then chemically labelled using the Label IT nucleic acid labelling kit (Mirus Bio, Madison, WI).
Hybridization protocol
Slides were pre-hybridized for 1 h at 37°C with a DIG easy hybridization solution (Roche Applied Science, Laval, QC, Canada) containing 5% BSA. Samples were then assigned randomly to a sub-array and hybridization was carried out for approximately 20 h at 37°C on a Slide Booster apparatus (Implen, Calabasas, CA). Slides were washed three times for 5 minutes at 37°C in 0.1X SSC / 0.1% SDS followed by a single wash for 5 minutes at 37°C in 0.1X SSC.
Scan protocol
Slides were scanned in a ScanArray GX PLUS Microarray Scanner (Perkin-Elmer, Boston, MA) at a resolution of 10µm
Description
n/a
Data processing
Spot presence-absence was scored by visual examination of the gridded microarray images. If a spot was visible by eye, it was scored as present (1), if not it was scored as absent (0). For a taxon to be scored as present, all of its triplicate spots had to be scored as present. The results from the 16S rRNA gene microarray were only used in the binary form (presence/absence of taxa).
