( Plates were transferred to ...)Monitoring Microbial Populations Using Real-Time qPCR on the MJ Research Opticon 2 System

Plates were transferred to the DNA Engine Opticon 2 system (MJ Research) following reaction assembly. Amplification was performed in calculated control mode according to the cycling program listed in Table 2. The optimal primer-annealing temperature was determined experimentally using the temperaturegradient feature of the DNA Engine Opticon 2 system. Melting curve analysis was performed to assess reaction specificity and to distinguish amplification products. The temperature at which fluorescence was measured during each cycle was set approximately 3C below the T_m of the amplicon to minimize any signal from non-specific amplification.⁸

Nested primer sets were used for the two rounds of PCR. For the first round, a 672bp fragment from amoA target gene was amplified using the primers: forward, 5'-GGN-GAC-TGG-GAC-TTC-TGG-3', and reverse, 5'-CCC-CTC-KGS-AAA-GCC-TTC-TTC-3'. (N = A, C, G, or T; K = G or T; S = C or G). For the second round of PCR the following primers amplified a 491bp amplicon from the first round PCR product: forward, 5'-GGG-GTT-TCT-ACT-GGT-GGT-3', and reverse, 5'- CCC-CTC-KGS-AAA-GCC-TTC-TTC-3'.

As an internal control, a plasmid DNA template (pUC19 from Invitrogen Corp.) was mixed with 0.25l of DNA extracted from each soil sample, and was subjected to the real-time qPCR protocol designed for amoA (Table 2). This method controls for possible interference and PCR inhibition from substances in the soil DNA samples. The following primer set was used to amplify a 200bp amplicon from the pUC plasmid in both the primary- and the nested-PCR reactions: forward, 5'-GTA-AAA-CGA-CGG-CCA-G-3', and reverse, 5'-CAGGAA- ACA-GCT-ATG-AC-3'.

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