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Monitoring Microbial Populations Using Real-Time qPCR on the MJ Research Opticon 2 System

plants. The first step of nitrification, the oxidation of ammonia to hydroxylamine, is catalyzed by amoA, the alpha subunit of ammonia monooxygenase found only in ammonia-oxidizing bacteria.5

To quantitate AOB populations, we developed a real-time qPCR assay for the amoA gene that uses SYBR Green I as an indicating fluorophore.6 Levels of amoA were determined for samples from each of sixteen soiltreatment groups. Each group was exposed to a different combination of two levels of each of the four environmental factors described above. A full report of this research has been submitted for publication.7


Materials and Methods
Experimental facility. The JRGC research facility and complete experimental design for this study are described in references 4 and 7.

DNA Extraction from Soil
Four soil cores from each subplot were extracted aseptically to a depth of 15cm with a 2.2-cm diameter corer. The cores were taken in late April, at the end of the second growing season of the experiment. The UltraClean Soil DNA Kit (Mo Bio Laboratories, Solana Beach, CA) was used to extract total DNA from a 0.5g sub-sample of the consolidated cores.

Real-Time qPCR
All DNA samples were analyzed in triplicate. Volumes of individual components and final reaction concentrations are listed in Table 1. SGI was from Molecular Probes (S-7567). MasterAmp PCR premix F was from Epicentre Technologies (MHF-925F). AmpliTaq was from Applied Biosystems (4338856). Reaction components were assembled in white-shell/white-well Hard-Shell microplates (MJ Research #HSP- 9655) and sealed with ultra-clear strip caps (MJ Research TCS-0803). '"/>

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