IMIS - Marine Research Groups

Study Extent: Samples were taken from lakes in the Vestfold Hills (East Antarctica) across a salinity gradient spanning from 0 to 250 psu, and over the austral summer period (December 2008-February 2009).

Method step description:

1. Community DNA from each sample was extracted from filter sections using a modified CTAB (Cetyl trimethyl ammonium bromide) extraction protocol in 2mL microfuge tubes along with 0.15g of beads from the PowerSoil™ DNA isolation kit (MOBio, California, USA). Each sample was incubated for 45min at 65°C in a mix of 700μL of CTAB, 24 μL β-mercaptoethanol, and 0.4 mg/mL of RNaseA (Sigma-Aldrich Sweden AB, Stockholm, Sweden), mixing by vortex agitation every 15 min. Samples were then ice-shocked and purified in 700μL of Chloroform:Isoamyl (24:1). After centrifugation (14,000g, 10 min, 4°C), the upper phase was removed and stored at -20°C for 60 min in one volume of isopropanol and a 0.5 volume of NaCl 5M. DNA was then precipitated through two consecutive centrifugations, first at14,000 g and 4°C during 30 min and afterwards, using the same setup during 15 min with 400 μL of ethanol 75%. After removing the supernatant, DNA pellets were dried using a speed vacuum DNA dryer at 300 g, 43°C, during 20 min. Pellets were then dissolved in 40-100 μL of milliQ water. The quality and quantity of DNA was measured using agarose gels (1.5%, TBE 0.5x) as well as a Nanodrop 2000/2000C Spectrophotometer v1.0 (Thermo Scientific, NanoDrop products, Delaware, USA).
2. Polymerase chain reactions (PCR) were performed in 96 wells plates. For each template DNA, four to nine replicate PCRs were performed following Logares et al. (2012), and subsequently combined. The PCR mix comprised 12.5 μL of 2x Phusion® GC Master Mix (F-532L, Finnzymes Oy, Vantaa, Finland), 4% of DMSO, 10 pmol μL-1 of barcoded fusion primers (Invitrogen™, Life Technologies Europe BV, Stockholm, Sweden), and 5 ng of DNA template in a final volume of 25μL that was adjusted with distilled water. Universal primers adapted from Stoeck et al. (2010) were used to amplify fragments of about 380bp from the V4 18S rDNA region and subsequently sequenced using 454 pyrosequencing. Primers included adapters for uni-directional (library Lib-L) sequencing. The forward primer comprised, in the 5’-3’ direction, a 454-specific adapter, followed by a multiplex identifier (MID, 7bp) and the template-specific primer. The reverse primer included a 454-specific adapter and the template-specific primer. PCR amplification, including number of cycles, was performed as described in Logares et al (2012). Quality control and quantification of PCR products were performed using agarose gels (1.5%, TBE 0.5x). PCR products were purified using the QIAquick® PCR purification Kit (QIAGEN GmbH, Hilden, Germany) and eluted in 45 µl of water (molecular biology grade). Amplicon concentration was estimated using Nanodrop (Thermo Scientific, Wilmington, USA).
3. 454-pyrosequencing was conducted at the Lund University Sequencing Facility. Two pools of fourteen samples, containing 333.3 ng of DNA each, were produced. In each pool, short fragments were removed using magnetic beads (Agencourt AMPure XP; Beckman Coulter AB, Bromma, Sweden). Pools were subsequently inspected using a DNA 1000 kit on a 2100 Bioanalyzer (Agilent Technologies Sweden AB, Kista, Sweden). Amplicons were quantified using the Quant-iT dsDNA assay kit (Invitrogen) and a Quantiflour fluorometer (Promega Biotech AB, Nacka, Sweden). Pools were diluted to obtain a total of 1×107 copies µL-1. Titration and library production (aiming at 10-15% recovery) were performed using emulsion PCR with the Lib-L kit (Roche AB, Stockholm, Sweden) for unidirectional sequencing. DNA positive beads were enriched, counted on an Innovatis CASY particle counter (Roche), processed using the XLR70 sequencing kit (Roche), and loaded onto a picotiter plate for pyrosequencing on a GS FLX Titanium platform (Roche).