Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Quantification of DNA by Real-Time PCR

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Before running a real-time PCR experiment, it is important to optimize the concentration of the primers (and probe, if using TaqMan chemistry) and determine the efficiency, sensitivity, and reproducibility of the assay (see Protocols 1 and 2). When designing a real-time PCR experiment, it is important to consider the type of quantitation (data analysis) method that will be used (see the section Extracting Data from a Real-Time PCR Experiment in the chapter introduction). If absolute quantification will be used, a standard curve must be run in parallel with the test samples (see Protocol 2). The standard curve may use plasmid DNA or other forms of DNA in which the absolute concentration of each standard is known. One must be sure, however, that the efficiency of PCR is the same for the standards as for the unknown samples. If either of the relative methods of quantification will be used, an endogenous reference gene must also be analyzed in parallel with the test samples (see the chapter introduction).


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Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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