Bacterial diversity and successional patterns during biofilm formation on freshly exposed basalt surfaces at diffuse-flow deep-sea vents- Panel9C
Primers 517F and 806R were used to target the V4 region of the 16S rRNA gene (Caporaso et al., 2010).  For multiplex sequencing, 8 forward primers were synthesized (Eurofin), each with a different tag. PCR reactions (50 µl volume) contained 250 nM of each of forward and reverse primers, 5 ng of template DNA and were performed using Picomaxx taq polymerase (Invitrogen, Carlsbad, CA) using the thermal profile 95°C for 2 min followed by 25 cycles of denaturation at 94°C for 15 s, primer annealing at 61°C and extension at 72°C for 45 s, with final extension of 72°C for 3 min. Amplicons were sequenced at Selah Genomics using 454 FLX chemistry.