Preliminary analysis of a former subglacial lake sediment in Antarctica
Direct culture     Samples of sediment (~0.1 g) were aseptically added to 10 mL  dd H2O and shaken until the sediment was fully dispersed. Using a pipette, 200µL of the suspension was removed and spread evenly on the surface of agar plates. Agar plates of the following types were prepared: 0.001, 0.01 and 0.1% wt/vol tryptone soya broth (Oxoid, Basingstoke, UK). 0.001 and 0.01% wt/vol yeast extract (Oxoid, Basingstoke, UK) and 0.001 and 0.01% yeast extract in a minimal salts medium [30]. The pH of all plates was 7-7.5. All plates were made with 2% agar (Oxoid, Basingstoke, UK; Bacteriological Agar No. 1), which was washed according to the method of Ryan [31]. Plates were incubated under aerobic conditions at 21 and 4°C for three months and observed during this time for colony formation.      DNA extraction and PCR amplification     Samples of frozen surface sediment (~0.5g) were removed from sediment cores for DNA extraction. DNA extractions were carried out using the PowerSoil DNA isolation kit (Mo Bio, California, USA) following the manufacturers standard protocol. The resulting DNA was quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA). Each extraction from 0.5g sediment produced ~250ng of DNA in 100µl nuclease free water.   16S amplification was carried out on sediment DNA obtained from the four depth sections A-C (15ng input DNA) using  787f ATTAGATACCCNGGTAG and 1492Rm GNTACCTTGTTACGACTT primers at an annealing temperature of 50°C to produce an amplicon of 705bp. PCR reactions were 15µl and contained 1X PCR buffer, 2.0 mM MgCl2, 0.25 mM dNTPs, 0.25 mM each primer, 0.4 Units BioTaq (Bioline, London, UK) and c. 15 ng template DNA. For Thermocycling: 1 X 95˚C for 50 s; 30 X 95˚C for 20 s, 50˚C for 30 s, 72˚C for 3 mins (ramp 72˚C at 0.3˚C/sec); 1 X 72˚C for 10 mins. Multiple PCR amplifications (24 in total) were cleaned and pooled to produce 900ng of 16S enriched DNA for 454 library preparation.    454 Pyrosequencing    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, 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).  The library was then subjected to clonal amplification by emulsion PCR followed by pyrosequencing on a 454 GS FLX sequencer (Roche) according to the manufacturers instructions (NEB NextQuick 454 library prep kit E6090).