16S rRNA amplicon sequencing of a microbial community derived from lake water
DNA was extracted using the Powerlyzer Powersoil kit (MoBio; Carlsbad, USA) following the manufacturer’s protocol with a few changes. 500 µl of the thawed plate wash was centrifuged for 5 minutes at 10.000 x g. the Supernatant was removed and the pellet dissolved by adding 750 µl bead solution and vortexing. The Suspensions were transferred to Glass Bead Tubes, 60 µl C1 solution was added and samples were placed in a Bead Beater for 5 minutes at 2000 RPM. Hereafter the manufacturer’s protocol was followed.  DNA concentrations were measured on Qubit 2.0 (Life Technologies, Invitrogen; Carlsbad, USA) and stored at -80°C until sequencing. The extracted DNA was PCR-amplified using the universal primer set PRK341F (5'- CCTAYGGGRBGCASCAG-3') and PRK806R (5'-GGACTACNNGGGTATCTAAT-3') that amplify the V3-V4 hypervariable regions of the 16S rRNA genes (56). 2 x 300 bp Purified PCR products were sequenced on the Illumina MiSeq platform at the DTU Multi Assay Core Center (Lyngby, DK).