Use of PCR-DGGE Based Molecular Methods to Analyse Microbial Community Diversity and Stability during the Thermophilic Stages of an ATAD Wastewater Sludge Treatment Process as an Aid to Performance Monitoring
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′.