16SrRNA of microcosm enrichment, solid substrates: source drill hole QV1,3 anaerobic treatment
Region-specific primers developed by the Earth Microbiome Project were used to amplify the V4 region (515F: GTGCCAGCMCCGCGGTAA and 806r: GGACTACHVGGGTWTCTAAT) of the 16S rRNA (rDNA) gene. Amplicons were generated in 25_L polymerase chain reactions (PCR) containing 12_L of MoBIO PCR Water (MoBIO Laboratories, Inc., Carlsbad, CA, USA), 10_L of 5 PRIME HotMasterMix (5 PRIME, Inc., Gaithersburg, MD, USA), 1_L forward primer (5_M concentration), 1_L of Golay barcode-tagged reverse primers (5_M concentration), and 1_L of template (sample) DNA. Reactions were run at 94¡C for 3 minutes to denature DNA, followed by 35 cycles of the following: 94¡C for 45 seconds, 50¡C for 60 seconds, and 72¡C for 90 seconds, with a final extension time of 10 minutes at 72¡C. The reaction was run in triplicate, and the results were pooled. These were cleaned using the UltraClean¨ PCR Clean-Up Kit (MoBIO) and loaded onto the Illumina MiSeq using custom sequencing primers and procedures (Caporaso et al., 2012).