|
| Status |
Public on Jan 30, 2015 |
| Title |
B3 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Universal Internal Standard + 70-mer
|
| Organism |
synthetic construct |
| Characteristics |
molecule type: 70-mer probe conjugated to the 20-mer standard DNA oligo
|
| Extracted molecule |
other |
| Extraction protocol |
DNA was extracted from the filters using the PureGene DNA kit (Gentra, Minneapolis, MN) or the AllPrep DNA/RNA Mini Kit (Qiagen Sciences, Maryland, USA) with slight modifications (as in Ward 2008).
|
| Label |
Cy5
|
| Label protocol |
PCR amplification using the nirS1F and nirS6R primers of Braker et al (1998). The PCR fragments were labeled with amino-allyl-dUTP (Ambion) during linear amplification using random octamers and a Klenow polymerase (Applied Biosystems). The reaction contained 3.96 mM d(AGC)TP, 0.44 mM dTTP, and 4.84 mM dUaa and was amplified for 3 h at 37°C. The Klenow product was purified and conjugated with Cy3. The Cy3-labelled target (200 ng) was combined with 2x hybridization buffer (1x final concentration; Agilent) and 0.25 pmol of a Cy5-labelled complementary 20-mer standard oligonucleotide and incubated at 95°C for 5 min before being cooled to room temperature.
|
| |
|
| Channel 2 |
| Source name |
marine sediment
|
| Organism |
aquatic metagenome |
| Characteristics |
location: Mesocoxm H1 day 3
location coordinates: station CT2 in Chesapeake Bay, 38°37.191′ N 76°08.061′ W
molecule type: PCR fragment, nirS gene from denitrifying bacteria amplified from DNA extracted from marine sediments
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from the filters using the PureGene DNA kit (Gentra, Minneapolis, MN) or the AllPrep DNA/RNA Mini Kit (Qiagen Sciences, Maryland, USA) with slight modifications (as in Ward 2008).
|
| Label |
Cy3
|
| Label protocol |
PCR amplification using the nirS1F and nirS6R primers of Braker et al (1998). The PCR fragments were labeled with amino-allyl-dUTP (Ambion) during linear amplification using random octamers and a Klenow polymerase (Applied Biosystems). The reaction contained 3.96 mM d(AGC)TP, 0.44 mM dTTP, and 4.84 mM dUaa and was amplified for 3 h at 37°C. The Klenow product was purified and conjugated with Cy3. The Cy3-labelled target (200 ng) was combined with 2x hybridization buffer (1x final concentration; Agilent) and 0.25 pmol of a Cy5-labelled complementary 20-mer standard oligonucleotide and incubated at 95°C for 5 min before being cooled to room temperature.
|
| |
|
| |
| Hybridization protocol |
Samples were hybridized to single or duplicate arrays by overnight incubation at 64°C and washed.
|
| Scan protocol |
The arrays were scanned with a laser scanner (Molecular Devices 4300)
Images were analyzed with Gene Pix Pro 6.0 software (Molecular Devices)
|
| Description |
The same sample was hybridized to two arrays for use as replicates.
|
| Data processing |
Quality control was employed to remove noise. For each channel, i.e. 532 nm (Cy3) and 635 nm (Cy5), the average background fluorescence was recalculated after excluding background fluorescence values greater than the upper whisker of all of the background fluorescences. This limit was defined as the 75th percentile plus 1.5 times the interquartile range.
Cy3/Cy5 fluorescence intensities were standardized to the highest Cy3/Cy5 fluorescence across the AOA probe set (normalized fluorescence ratio; FRN). The data for each sample were then averaged among replicates except when one replicate was zero, in which case the zero value was ignored.
|
| |
|
| Submission date |
Jan 29, 2015 |
| Last update date |
Jan 30, 2015 |
| Contact name |
Bess B Ward |
| E-mail(s) |
bbw@princeton.edu
|
| Phone |
6092585150
|
| Organization name |
Princeton University
|
| Department |
Geosciences
|
| Lab |
Guyot Hall
|
| Street address |
Washington Road
|
| City |
Princeton |
| State/province |
NJ |
| ZIP/Postal code |
08544 |
| Country |
USA |
| |
|
| Platform ID |
GPL19719 |
| Series (1) |
| GSE65430 |
Organic matter loading modifies the microbial community responsible for nitrogen loss in estuarine sediments |
|
