Table 3.
Optimized conditions for DGGE analysis.
Amplicon | Primers set | Optimal parameters (DGGE analysis) | |||||
---|---|---|---|---|---|---|---|
Amount of DNA template in PCR reaction | Voltage, V | Time, hrs | Denaturant range, (%) | Amount of amplicon (per lane), μg | Gel staining methods | ||
V3–V5; 16S rRNA | 338F-GC*/518R | 300 ng | 75 | 16 | 35–75 | 4.5 | EtBr |
V6–V8, 16S rRNA | 948F-GC*/L1401 | 300 ng | 75 | 18 | 40–65 | 1.5 3 |
Silver stain EtBr |
rpoB | 1698F-GC*/2043R | 600 ng | 75 | 16 | 30–60 | 0.3 | EtBr, silver stain |
*GC clamp added to the 5′ end of the primers 338F, 948F, and 1698F, 5′CGCCCGCCGCGCGCGGCG GGCGGGGCGGGGGCACGGGGGG-3′.