T-RFLP Analysis of 16S rRNA and mcrA Amplicons

The T-RFLP analysis of bacterial and methanogenic communities using FAM-labeled PCR products was done as described previously (Sträuber et al., 2012; Lucas et al., 2015). PCR product quality was checked by agarose gel electrophoresis and amplicons were purified with SureClean (Bioline, Luckenwalde, Germany). Purified PCR products were quantified after electrophoresis in 1.5% agarose gels with ethidium bromide staining using the GeneTools program (Syngene, Cambridge, UK). The purified PCR products were digested with restriction endonucleases purchased from New England Biolabs (Schwalbach, Germany). The mcrA amplicons were digested with MwoI and the 16S rRNA amplicons with RsaI, using 2 units of the respective enzyme for digesting 10 ng of PCR product at 37°C overnight. The subsequent T-RFLP analysis was done for the mcrA amplicons with the GeneScan^TM-500Rox^TM (Applied Biosystems, USA) as fragment size standard and for the 16S rRNA amplicons with the MapMarker1000 (BioVentures Inc., USA). Resulting electropherograms were analyzed by using the GeneMapper 5 software (Applied Biosystems) and processed according to Abdo et al. (2006). To differentiate between peaks and background, signals with low peak areas were removed according to eight times the standard deviation.