Dataset: qPCR data for archaeal amoA ecotypes
Deployment: KM1128

Nucleic acids (DNA) were extracted as described previously (Santoro et al. 2010), with slight modifications. Briefly, cells on the filters were lysed directly in the bead beating tubes with sucrose-ethylene diamine tetraacetic acid (EDTA) lysis buffer (0.75 M sucrose, 20 mM EDTA, 400 mM NaCl, 50 mM Tris) and 1% sodium dodecyl sulfate (SDS). Filter samples were subject to three freeze-thaw cycles of 5 min in liquid nitrogen and 5 min in a 65 degree C water bath. Tubes were then agitated in a bead beating machine (Biospec) for 1.5 min, and proteinase K (Invitrogen) was added to a final concentration of 0.5 mg mL-1. Filters were incubated at 55 degrees C for approximately 4 h and the resulting lysates were purified with the DNeasy kit (Qiagen) using a slightly modified protocol (Santoro et al. 2010). The purified nucleic acids were eluted in 200 uL of DNase, RNase-free water (Gibco) and quantified using a fluorometer (Qubit and Quanti-T BR reagent, Invitrogen Molecular Probes).

All qPCR assays were conducted using group-specific assays for the thaumarchaeal amoA gene for 'shallow' water column ecotype A (WCA) and 'deep' water column ecotype B (WCB) (Mosier and Francis 2011) with TaqMan Environmental Mastermix (Life Technologies) chemistry on a CFX96 qPCR machine (Bio-Rad, Inc., Hercules, CA). Detection limits for TaqMan assays were 1 copy mL-1 or better. All samples were run in triplicate against a standard curve spanning approximately 101 - 105 templates, run in duplicate. Plasmids containing cloned inserts of the target gene (TOPO pCR4 vector, Invitrogen or pGem vector, Promega) were used as standards. Standards were linearized with the restriction enzyme NotI (New England Biolabs), purified (DNeasy, Qiagen), quantified by fluorometry (Quanti-T HS reagent, Invitrogen), and stored at -80 degrees C. Fresh standard dilutions were made from frozen stocks for each day of analysis. qPCR was carried out using the following thermal profile: 95 degreesC for 10 min, followed by 40 cycles of 95 degrees C for 30 s and 55 degrees C for 30 s. A minimum of three negative control qPCR reactions to which no DNA template was added were run with every assay.

Gene copies per qPCR reaction were converted to volumetric concentrations (i.e. copies mL-1 seawater) using the volume of seawater filtered, the DNA elution volume, and the volume of purified DNA used per reaction, and are reported as the mean of triplicate analyses.

BCO-DMO Processing:
- re-formatted date and time; added ISO date/time field;
- copied station, date, time, lon, and lat from separate spreadsheet into dataset.