171 days of incubation under methane atmosphere with no HPG added: Sample 7135-171d-CH4-noHPG
"Sediment DNA was extracted using the Power Soil DNA Isolation Kit according to the manufacturer's protocol (MoBio, Carlsbad, CA) and diluted DNA from genome-amplified sorted consortia was used directly. The V4 region of the 16S rRNA gene was amplified from each extract using archaeal and bacterial primers 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT) (Caporaso et al., 2011; Caporaso et al., 2012). Sediment samples were amplified in duplicate. The non-barcoded primers were used with Q5 Hot Start High-Fidelity 2x Master Mix (New England Biolabs, Ipswich, MA ) according to the manufacturer's directions using annealing conditions of 54°C for 30 cycles and 58°C for 32 cycles for sediments and MDAs, respectively. Duplicates of sediment sample amplifications were then pooled. The barcoded 806R primer (CAAGCAGAAGACGGCATACGAGAT XXXXXXXXXXXX AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT) was paired with 515F in a reconditioning reaction (same conditions as above except for 5 cycles of PCR) to barcode the PCR products. Samples were mixed together in equimolar amounts and purified in bulk through a Qiagen PCR purification kit before submission to Laragen (Culver City, CA) for analysis on an Illumnia MiSeq platform. The resulting paired-end sequence data, 2x 250 base pairs (bp), was de-multiplexed and sequences with >1 bp mismatch on the 12 bp barcode were removed. The resulting sequences were passed through Illumina’s MiSeq Recorder software to assign quality scores to each base call and remove adapter, barcode and primer sequence."
California Institute of Technology
7135-171d-03-CH4-noHPG