Denaturing gradient gel electrophoresis (DGGE) of amplified 16S rRNA genes

PCR primers 968F/1401R were used to amplify the V6-V8 portion of 16S rRNA genes as previously described²⁶. Archaea- and fungus-specific primers were used to amplify archaeal 16S and fungus ITS genes, respectively (Supplementary table S1)^42,43. DGGE analysis was performed using the Bio-Rad DCode system. Electrophoresis was done using a 16 × 16 cm, 1 mm thick gel that contained 8% polyacrylamide with a 20 to 80% denaturant gradient (100% denaturant was 7 M urea and 40% (v/v) deionized formamide). The gels were run at 100 V for 16 h at 60 °C in TAE buffer (40 mM Tris–acetate, 1 mM EDTA; pH 7.4). After electrophoresis, the gels were stained for 30 min in TAE buffer with SYBR-Gold nucleic acid gel stain (S-11494, Invitrogen) for photographing. Gels were scanned using a GS-800 calibrated densitometer (Bio-Rad). Analysis of DGGE profiles (band match and clustering) was carried out using Quantity One software (version 4. 6.1; Bio-Rad), as described previously⁴⁴.