IMIS - Marine Onderzoeksgroepen

Samples were drawn from 10 and 100 m depths from the northern and southern, inshore and offshore corners of the Palmer LTER sampling grid. We collected duplicate samples using a rosette equipped with 10 l Niskin bottles and conductivity, temperature, and depth (CTD) probes. To contrast summer and winter water, we also collected an additional Austral winter sample from 10 m depth at the northern, inshore sampling site in August 2008, using a submersible pump with silicone tubing. Water samples were filtered (1 to 2 l) through 0.2 μm SterivexTM filters (Millipore), preserved genomic DNA by flooding the 2 ml filter cartridge reservoir with sucrose lysis buffer (40 mM EDTA, 50 mM Tris-HCl, 0.75 M sucrose), and stored the filters at −80°C until processing.
Study Extent: Sampling was conducted on the annual Palmer LTER (western coast of the Antarctic Peninsula) midsummer research cruise (January−February 2008).
Method step description:

1. We extracted DNA using a Puregene DNA extraction kit (Qiagen), with modifications as described by Amaral-Zettler et al. (2009), and stored the DNA at −20°C until PCR amplification. Bacterial and archaeal V6 16S rRNA and eukaryotic V9 18S rRNA gene hypervariable regions were amplified as described previously (Huber et al. 2007, Amaral- Zettler et al. 2009), using ‘barcoded’ primers which al- lowed for multiplexed sequencing (see http://vamps. mbl.edu/resources/primers.php for details). For each sample, we pooled triplicate 50 μl PCR reaction products to minimize propagation of PCR errors and purified them using a QIAquick column-based purification kit (Qiagen). We sequenced purified amplicons on a 454 Genome Sequencer FLX (Roche) according to the manufacturer’s protocols using the LR70 kit.