Amplification and Fingerprinting of Microbial Communities

Total community DNA was used as the template for amplification of the partial 16S rRNA gene fragment using Taq DNA polymerase (Bioline, Lückenwalde, Germany) with primer F968 with a GC clamp attached to the 5′ end and universal bacterial primer R1401.1b. For ITS1 amplification, primers EF4/ITS4 were used; this PCR was followed by a second amplification with primers ITS1f-GCITS2. Primer sequences, the reactions mixtures, and cycling conditions have been described (Pereira e Silva et al., 2012 (link)). The DGGE was performed in 6 % (w/v) polyacrylamide gels with 45–65 % and 20–50 % denaturant gradients for bacterial and fungal communities, respectively (100 % denaturant is defined as 7.0 M urea with 40 % deionized formamide). Electrophoresis was carried out at 100 V and 75 mA, for 16 h at 60 °C. The gels were subsequently stained for 40 min in 0.5 % TAE buffer with SYBR gold (final concentration 0.5 μg/L) (Invitrogen, Breda, the Netherlands) (Fig. S1 in the Supplementary Material). Gel images were digitized using Imagemaster VDS (Amersham Biosciences, Buckinghamshire, UK). The DGGE patterns were then transformed to a band-matching table using GelCompar II software (Applied Maths, Sint Martens Latem, Belgium).