IMIS - Marine Onderzoeksgroepen

A 3.8 m sediment core was extracted at a depth of 93.4 m below the ice surface inside a virkon sterilized and polycarbonate lined core barrel. When taken all core samples remained within the core liners and were immediately frozen until sub-sampling in a clean class II microbiological safety cabinet in Cambridge. Gloves were used at all times and instruments used in laboratory manipulations were autoclaved.
Study Extent: Lake Hodgson, Antarctica
Method step description:

1. Samples of frozen surface sediment (~0.5 g) were removed from sediment cores for DNA extraction. DNA extractions were carried out using the PowerSoil DNA isolation kit (Mo Bio, Carlsbad, CA, USA) following the manufacturers standard protocol. The resulting DNA was quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Each extraction from 0.5 g sediment produced ~250 ng of DNA in 100 μL nuclease free water. 16S RNA gene amplification was carried out on sediment DNA obtained from the four depth sections A–C (15 ng input DNA) using forward primer 787f ATT AGA TAC CCN GGT AG and reverse primer 1492Rm GNT ACC TTG TTA CGA CTT at an annealing temperature of 50 °C to produce amplicons of 705 bp. PCR reactions were 15 μL and contained 1× PCR buffer, 2.0 mM MgCl2, 0.25 mM dNTPs, 0.25 mM each primer, 0.4 Units BioTaq (Bioline, London, UK) and ca. 15 ng template DNA. For Thermocycling: 1 ×95 °C for 50 s; 30 ×95 °C for 20 s, 50 °C for 30 s, 72 °C for 3 min (ramp 72 °C at 0.3 °C s−1); 1 ×72 °C for 10 min. Multiple PCR amplifications (24 in total) were cleaned and pooled to produce 900 ng of 16S enriched DNA for 454 library preparation.
2. An amplicon library was generated using the Rapid Library Preparation kit and following the manufacturer’s recommendations in the GS FLX Titanium Series Rapid Library Preparation Method Manual (Roche). Briefly, the PCR amplicons were purified using AMPure beads (Agencourt Bioscience Corporation, Beverly, MA, USA), adaptors were blunt-end ligated to the fragment and the dsDNA amplicon library was quantitated via fluorometry using Quanti-iT Pico Green reagents (Invitrogen, Carlsbad, CA, USA). The library was then subjected to clonal amplification by emulsion PCR followed by pyrosequencing on a 454 GS FLX sequencer according to the manufacturer’s instructions (NEB NextQuick 454 library prep kit E6090).