IMIS - Marine Research Groups

Thirteen samples were taken at 25 m depth (the layer of maximum oxygen), three samples were taken at 2 m and two samples were taken at 50 m depth. An SBE 911 plus CTD instrument combined with an SBE 32 Carousel water sampler (both by Sea-Bird Electronics) equipped with 24 Niskin bottles was used to collect seawater and measure physical parameters (temperature and salinity). +- 2 L of seawater was pre-filtered through 3 μm pore size polycarbonate membranes (Whatman), and then filtered with a vacuum pump through polycarbonate membranes (47 mm diameter, 0.2-μm pore size, Whatman). Samples were subsequently frozen at −80°C.
Samples (n=18) were collected in the waters by the northern tip of the Antarctic Peninsula, including the northern area of the Bransfield Strait, the Powell Basin and the South Orkney tableland area during the 28th Chinese National Antarctic Research Expedition, from December 2011 to January 2012.
The PCR products were purified using AxyPrepTM DNA Purification Kit (Axygen®) and quantified using a Qubit® 2.0 Fluorometer (Life Technologies).
DNA was extracted using a modified cetyltrimethylammonium bromide method and examined by agarose gel electrophoresis.
The V1–V3 region of the 16S rRNA gene of Bacteria was amplified using the universal primer pair F8 (5′-CCTATCCCCTGTGT- GCCTTGGCAGTCTCAG-AGAGTTTGATCCTGGCTCAG-3′) and R533 (5′-CCATCTCATCCCTGC- GTGTCTCCGACTCAG-NNNNNNNN-TTACCGCGGCTGCT- GGCAC-3′; NNNNNNNN being the place of the sample-specific barcode). PCR was performed using 5–10 ng genomic DNA in a final volume of 50 μL. The PCR procedure was as follows: initial denaturation at 95°C for 2 min; 25 cycles at 95°C for 30 s, 56.4°C for 1 min and 72°C for 30 s; and a final extension at 72°C for 5 min.
454 pyrosequencing was performed on an FLX Titanium Genome Sequencer (454/Roche Life Sciences).