IMIS - Marine Onderzoeksgroepen

A discrete water sample for nucleic acid extraction was collected in situ at 850 m with a Large Volume Water Transfer System (WTS-LV; McLane Research Laboratories, East Falmouth, Massachusetts). This system was fitted with a stacked 143 mm diameter filter housing that allowed the sample to be size fractionated into 10 um, 3 um, and 0.2 um classes (Supor Membrane filters were used; Pall). This system allowed us to concentrate 295 L of seawater on 142 mm filters during a 4 h in situ deployment. An additional discrete water sample (300 mL) from 30 m was obtained for nucleic acid extraction from a 10 L Niskin bottle sample that was fil- tered through a 47 mm 0.2 um Supor membrane filter (Pall). Filters from the WTS-LV were preserved as described previ- ously (Christner et al. 2014) while the entire 47 mm filter from the 30 m sample was placed in a 7 mL cryovial and preserved with 7 mL of a solution of 40 mM EDTA pH 8.0, 50 mM Tris pH 8.3, and 0.73 M Sucrose to prevent cell lysis during storage.
Study Extent: After drilling through the Ice sheet (56m), water samples were taken from the marine cavity beneath the McMurdo Ice Shelf, Antarctica.
Method step description:

1. Nucleic acids were extracted from the 10 um, 3 um, and 0.2 um filters from 850 m and the 0.2 um filter from 30 m using a MO BIO PowerWater DNA Isolation Kit according to the manufactures instructions. The V4 hypervariable region of the small subunit (SSU) rRNA gene was amplified using the primers 515F and 806R (Caporaso et al. 2012) and sequenced on an Illumina MiSeq as described by Christner et al. (2014).