Incorrect parameter settings for the PCR machine will affect the results of PCR experiments, mainly in temperature, time and number of cycles. If the temperature parameters are set too high and the extension time is not enough, the experimental results will be affected.

Precautions for setting parameters of PCR thermal cycler:

1. Temperature and time

Temperature and time settings: Based on the three steps of PCR principle, three temperature points of denaturation, annealing and extension are set. The three-temperature point method is used in the standard reaction, and the two-temperature point method can be used for shorter target genes.

1. Denaturation temperature and time

Low denaturation temperature and incomplete melting are the main reasons for PCR failure.

2. Annealing (refolding) temperature and time

Annealing temperature is an important factor affecting PCR specificity.

3. Extension temperature and time

The extension temperature of the PCR reaction is generally selected between 70 and 75°C. Excessively high extension temperature is not conducive to the binding of primers and templates. The time of the PCR extension reaction can be determined according to the length of the fragment to be amplified. For amplification of low-concentration templates, the extension time should be slightly longer.

2. Number of cycles

The number of cycles determines the degree of PCR amplification. The number of PCR cycles mainly depends on the concentration of template DNA, generally between 30 and 40 times. The higher the number of cycles, the greater the amount of non-specific products.

According to the purpose of DNA amplification and detection standards, PCR machines can be divided into four categories: ordinary PCR machines, gradient PCR machines, in-situ PCR machines, and real-time fluorescence quantitative PCR machines.