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Calibration Parameters of Thermal Cycler (PCR)
2021-03-27 09:39
PCR amplification instrument is also called PCR gene amplification instrument, PCR nucleic acid amplification instrument, polymerase chain reaction nucleic acid amplification instrument, commonly known as PCR instrument. To put it simply, the content of the collected sample is too low to be detected and analyzed with the common instruments, so the PCR technology and the PCR instrument is needed to realize the sample amplification. The amplification factor is 2N power. Through this technology, the PCR instrument can be used for molecular biology research: nucleic acid quantitative analysis, gene expression difference analysis etc., and for medical research: prenatal diagnosis, pathogen detection and so on.
The reaction principle of PCR, which is also the working principle of the PCR instrument, includes three parts: high temperature denaturation, annealing renaturation, and extension and replication. One of the main parameters of the PCR machine is temperature, and a main factor that affects whether the temperature indication is accurate is the speed of heating. Temperature, as the basic setting for denaturation, renaturation, extension, is an item we need to calibrate.
During the reaction, we need to know whether the temperature indication and the set value are accurate. This is the first temperature indication error, because we need to increase temperature, decrease temperature, and then increase temperature, which involves a speed issue. The speed of raising and lowering the temperature will directly affect the entire reaction process. Therefore, the rate of raising and lowering the temperature is also a direction that we need to consider. The instrument uses the heating module to control the temperature. During the rapid temperature rise and fall, the instrument will inevitably increase/decrease the temperature before reaching the set temperature. This is the temperature overshoot, which is also an indicator to consider. After reaching a certain temperature, the instrument needs to be balanced for a period of time (the set time), and whether the temperature balance time is consistent with the set time is also a direction to be considered. Different DNA templates require different temperature and melting time. If the melting time does not meet the requirements and the DNA is not completely denatured, it will return to its natural state during the cooling and renaturation process.
The PCR machine has 48 holes, 96 holes, and 384 holes. Each hole can hold a sample. Naturally, the temperature parameters we mentioned above are still incomplete. Here is a rough summary of the calibration parameters of the PCR instrument: temperature indication error, temperature uniformity, maximum temperature impulse, temperature duration accuracy, average temperature rise and fall rate, maximum temperature rise and fall rate, etc.
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