12C-alginate amendment, replicate B, fraction 11
A 464-bp fragment of 16S rRNA genes was amplified using primers S-D-Bact-0341-b-S-17 (5'-CCTACGGGNGGCWGCAG-3') and S-D-Bact-0785-a-A-21 (5'-GACTACHVGGGTATCTAATCC-3'), in 12,5 l reactions containing 0.5 ng of DNA, 500 nM of each primer and 1X Master Mix Q5 (New England Biolabs). PCR was performed by heating reactions for 3 min at 95C, followed by 25 cycles of 30 s at 95C, 30 s at 55C, 30 s at 72C, and a final elongation step of 5 min at 72C. Amplicons were purified using AMPure XP beads (Beckman-Coulter, Brea, USA), attached to dual indices and Illumina adapters using the Nextera XT Index kit, analyzed on a Labchip GXT (Perkin Elmer, USA) and pooled at equimolar concentrations. The library was sequenced using a MiSeq Reagent kit v3 (2 x 300 cycles) at the Genomer Platform (Station Biologique de Roscoff, France).
12B-11