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Once primers and probes have been designed and obtained, it is necessary to optimize their concentration for each real-time polymerase chain reaction (PCR) assay. A set of PCRs is assembled… Click to show full abstract
Once primers and probes have been designed and obtained, it is necessary to optimize their concentration for each real-time polymerase chain reaction (PCR) assay. A set of PCRs is assembled in which the concentrations of forward and reverse primers are varied independently. Following amplification of the template DNA, amplification plots are compared. A standard curve is generated 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.
Journal Title: Cold Spring Harbor protocols
Year Published: 2018
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