Nucleic acid extraction and Denaturing gradient gel electrophoresis (DGGE)

Nucleic acids were extracted from 0,3 g of sieved sediment with a Power Soil DNA isolation kit (MoBio, Carlsbad, USA) according to the manufacturer’s instructions. 16S rRNA gene fragments (~350 bp) were amplified by PCR using primer pair specific for methanogens. Primer sequences are as follows, 0357 F-GC 5′-CCC TAC GGG GCG CAG CAG-3′ (GC clamp at 5′-end CGC CCG CCG CGC GCG GCG GGCGGG GCG GGG GCA CGG GGG G) and 0691 R 5′- GGA TTA CAR GAT TTC AC -3′ (Watanabe et al. 2004). PCR amplification was carried out in 50 pL reaction mixture contained within 0.2 mL, thin walled micro-tubes. Amplification was performed in a TC-XP thermal cycler (Bioer Technology, Hangzhou, China). The reaction mixture contained 5 pL of 10 * PCR amplification buffer, 200 pM of each dNTP, 0,8 pM of each primer, 8 pL of template DNA and 5.0 U of FastStart Taq DNA polymerase (Polymerase dNTPack; Roche, Germany). The initial enzyme activation and DNA denaturation were performed for 6 min at 95°C, followed by 35 cycles of 1 min at 95°C, 1 min at 55°C and 2 min at 69°C and a final extension at 69°C for 8 min (protocol by Watanabe et al. 2004). PCR products were visualised by electrophoresis in ethidium bromide stained, 1.5% (w/v) agarose gel.

DGGE was performed with an INGENYphorU System (Ingeny, Netherlands). PCR products were loaded onto a 7% (w/v) polyacrylamide gel (acrylamide: bisacrylamide, 37.5:1). The polyacrylamide gels were made of 0.05% (v/v) TEMED (N, N,N, N-tetramethylenediamine),

0. 06% (w/v) ammonium persulfate, 7 M (w/v) urea and 40 % (v/v) formamide. Denaturing gradients ranged from 45 to 60%. Electrophoresis was performed in 1*TAE buffer (40 mM Tris, 1 mM acetic acid, 1 mM EDTA, pH 7.45) and run initially at 110V for 10 min at 60°C, afterwatds for 16 h at 85 V. After electrophoresis, the gels were stained for 60 min with SYBR Green I nucleic acid gel stain (1:10 000 dilution) (Lonza, Rockland USA) DGGE gel was then photographed under UV transilluminator (Molecular Dynamics). Images were arranged by Image analysis (NIS Elements, Czech Republic). A binary matrix was created from the gel image by scoring of the presence or absence of each bend and then the cluster tree was constructed (programme GEL2k; Svein Norland, Dept. Of Biology, University of Bergen).