Polymerase Chain Reaction Amplification, Cloning, and Sequencing

The extracted DNA (mean 14 ng/μl) was used as template in PCR with pmoA primers A189F and mb661R (Costello and Lidstrom, 1999). This primer pair capture most groups of aerobic methane-oxidizing bacteria without amplification of equally abundant amoA genes (Bourne et al., 2001). PCR amplifications (for pmoA) were done in 20 μl reactions in 0.2 ml tubes using a thermal cycler (BioER). Each PCR mixture contained 2 mM MgCl₂, 1× PCR buffer (Invitrogen), 0.2 mM dNTPs, 0.25 μM each of the forward and reverse primers (A189F/mb661R), 4% bovine serum albumin (New England Biolabs, USA), 0.05 units of Taq DNA Polymerase-Recombinant (Invitrogen), and 10-fold diluted DNA. All PCR reactions were performed along with one negative control containing DNase/RNase free water instead of DNA template. A thermocycling program with an initial denaturation step at 94°C for 3 min followed by 40 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min, and a final extension at 72°C for 5 min was used. Primers used for PCR amplifications of pmoA, clone libraries and qPCR are listed in Supplementary Table S1. After amplification, 1% agarose gel electrophoresis was performed to verify correct amplification and size of the gene by comparison to a TrackIt 100 bp DNA Ladder (Invitrogen) (Supplementary Figure S2).

PCR products generated from DNA extracted from the bottom layer of two lakes (Ekoln and Ramsen), were cloned using the TOPO TA Cloning kit (Invitrogen) as previously described (Eiler and Bertilsson, 2004). Sanger sequencing was carried out at the Uppsala Genome Center using an ABI3730XL DNA Analyzer (Applied Biosystems). All nucleotide sequences were deposited at the National Center for Biotechnology Information (NCBI) under accession numbers {"type":"entrez-nucleotide-range","attrs":{"text":"KC588447-KC588466","start_term":"KC588447","end_term":"KC588466","start_term_id":"498541747","end_term_id":"498541785"}}KC588447-KC588466. A phylogenetic tree was constructed with Mega 5 using the maximum likelihood approach and Jukes-Cantor model. The topologies of the phylogenetic tree were calculated by bootstrap analysis with 1000 replications.