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

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Optimizing Primer and Probe Concentrations for Use in Real-Time PCR

(Protocol summary only for purposes of this preview site)

Once primers and probes have been designed and obtained (see the chapter introduction), it is necessary to optimize their concentrations for each real-time PCR assay. A set of PCRs is assembled in which the concentrations of forward and reverse primers are independently varied. Following amplification of the template DNA, amplification plots are compared. A standard curve is generated (Protocol 2) to determine the efficiency, sensitivity, and reproducibility of the assay. If SYBR Green I is used as the probe, then the melting curves are also analyzed.


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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