Optimized Cloning Protocol for Standard Curve Generation in qPCR of Environmental Functional Genes v1

Due to the complex and varied biochemical requirements and physiological behaviors of certain environmental bacterial and archaeal groups that carry specific functional genes as potential biomarkers, it is challenging to extract and maintain pure cultures of these organisms. Therefore, generating standard curves for qPCR necessitates cloning gene sequences from environmental DNA samples. This protocol was developed for cloning target genes from environmental samples to produce plasmid DNA, which was then used to prepare standard curves for quantifying functional genes, including mcrA, pmoA, amoA, and nosZ from water and soil samples. The procedure involves inserting amplified gene sequences into pGEM‱-T Vectors and transforming them into JM104 E. coli competent cells. Testing this protocol yielded higher amounts of pure plasmid DNA, making it suitable for standard curve preparation for qPCR of the target genes.