Sureclean plus kit

The alpha subunit of the methyl coenzyme M reductase (mcrA) gene was polymerase chain reaction (PCR) amplified using the mlas and mcrA-rev primer set and PCR program described earlier [42 (link)], which produces PCR products of approximately 500 bp. The mcrA-rev primer was 5’-labeled with the phosphoramidite fluorochrome, 6-carboxyfluorescein (FAM). The PCR products were purified using a SureClean plus kit (Bioline, Germany). The quality was checked by gel-electrophoresis using a 1.5% agarose gel and quantified with a NanoDrop ND-1000 UV. Following purification, the PCR products (10–30 ng per sample) were digested with BstNI (2 hours incubation at 60°C) and MwoI (37°C overnight) restriction enzymes. The digested PCR products were precipitated with 30 μL absolute ethanol and 2.5 μL EDTA, and the centrifuged pellet was washed in 70% ethanol. The washed and dried digested PCR products were resuspended in HiDi formamide with the fragment size standard GeneScan-500 ROX (Applied Biosystems GmbH, Weiterstadt, Germany). The fluorescently labeled terminal restriction fragments were separated via capillary electrophoresis with an automatic sequencer (ABI Prism 3130xl genetic analyzer; Applied Bio-systems, Weiterstadt, Germany). The taxonomy was assigned to the terminal restriction fragments using the database described in [43 (link)].

The plasmid DNA was subjected to overnight digestion at 37 °C with exonuclease V Rec BCD (New England Biolabs, Massachusetts, USA) to remove chromosomal DNA. PCR reactions using universal primers covering the V4 region of 16S rRNA gene were performed to check for chromosomal DNA contamination. The primers used for bacteria were F5′-CCTACGGGNGGCWGCAG-3′ (Bac_341F) and R5′-GGATTAGATACCCBDGTAGTC-3′ (Bac_785R)^{27 (link)}; and for archaea F5′-CCCTAYGGGGYGCASCAG-3′ (Arc_340F) and R5′-ATTAGAKACCCSNGTAGTCC-3′ (Arc_806R)^{28 (link)}. The plasmid DNA was purified using the SureClean Plus kit (Bioline, London, UK).