Fluorescence quantitative PCR instrument

Fluorescence quantitative PCR instrument is an important molecular biology instruments, the following for your detailed introduction:
Principle of operation
Fluorescence signal generation: Fluorescent groups, such as fluorescent dyes (SYBR Green Ⅰ, etc.) or fluorescent probes (TaqMan probe, etc.), are added to the PCR reaction system. The fluorescent dyes can be doped into the DNA double strand non-specifically and emit fluorescent signals; when the TaqMan probe is intact, the fluorescent signals emitted by the reporter group are absorbed by the quenching group, and when PCR amplification is performed, the 5′-3′ exonuclease activity of Taq enzyme enzymatically cleaves the probe to degrade it, so the reporter and quenching fluorescent groups can be separated, and fluorescence signals are thus generated. The fluorescent signal is generated.
Real-Time Monitoring: The PCR instrument detects the intensity of the fluorescent signal at the end of each cycle of the PCR reaction and records it in real time. As the PCR reaction progresses, the DNA copy number increases exponentially and the fluorescence signal increases.
Threshold setting and Ct value: Set a fluorescence threshold, generally use the fluorescence signal of the first 15 cycles of the PCR reaction as the fluorescence background signal, the default setting of the fluorescence threshold is 10 times the standard deviation of the fluorescence signal of 3-15 cycles. When the fluorescence signal of a sample exceeds this threshold, the corresponding number of cycles is the threshold cycle number (Ct value). The Ct value for each template is linearly related to the logarithm of the number of starting copies of that template, with the higher the number of starting copies, the smaller the Ct value.
Quantitative analysis: By comparing the standard curve with a series of standards of known concentration, the starting copy number of the unknown sample can be calculated from the standard curve according to the Ct value of the unknown sample, thus realizing the quantitative analysis of the unknown template.
Main components
Thermal cycling system: It can precisely control the temperature change of PCR reaction, including denaturation, annealing and extension steps, to ensure the smooth progress of PCR reaction.
Fluorescence Detection System: Used to excite fluorescent groups and detect the resulting fluorescence signals, with high sensitivity and stability, it can accurately measure the weak fluorescence changes and convert them into electrical or digital signals for recording and analysis.
Control system and software: The control system is responsible for coordinating the work of the thermal cycling system and the fluorescence detection system, realizing automated PCR reaction process control and data acquisition. The accompanying software is used to set experimental parameters, real-time monitoring of fluorescence signals, analyzing data, generating amplification curves and quantitative results, etc., which is convenient for users to operate and interpret the results.