O gene ruler

Amplified products were separated on 1.5% w/v agarose gel stained with ethidium bromide, in 0.5X TBE buffer. 2.5 μl of each amplified sample was loaded into each well of the electrophoretic tank (BioRad, USA) and left to run for 45 minutes at 100V. Gels were photographed under UV transilluminator. Sizes of the amplified products were determined using a 1 kb molecular weight marker (O’ gene ruler, Thermo scientific, USA) as standard.

We generated DGGE profiles using an Ingeny Phor-U® system (Ingeny International, Goes, the Netherlands). Amplicons (150 ng per lane) were loaded onto 6% (w/v) polyacrylamide gels in 0.5× Tris-acetate-EDTA (TAE) buffer, and we added the same reference marker to each gel for normalization purpose in computer analyses. We optimized our own marker for bacteria and used a 1 kb DNA ladder (O'Gene Ruler; Thermo Scientific, Vilnius, Lithuania) for fungi. Amplicons were run either in a 40–55% (bacteria) or in a 20–55% denaturant gradient (fungi), at 60°C for 16 h and at a constant voltage of 100V. After the run, gels were stained with SYBR gold (Molecular Probes, Leiden, the Netherlands) in 0.5× TAE buffer, in the dark for one hour. Then, we digitized DGGE profiles using a digital camera and stored as them TIFF files for computer analysis. Gels were normalized using the GELCOMPAR II software (Applied Maths, Sint-Martens-Latem, Belgium). After normalization, we compared community compositions by clustering lanes by Pearson's correlation coefficient implemented in the GELCOMPAR II software using the unweighted-pair group method with arithmetic mean, rolling-disk background subtraction, and no optimization (Rademaker et al. 1999 ; Kropf 2004 ).