Help: the reaction system evaporates after real-time fluorescence quantitative PCR. How to solve it? This is ABI 7500

Just because you close the lid doesn't mean you close the PCR tube. Maybe you use a domestic PCR tube. Usually, the quality of domestic products is slightly poor, and the seal itself is not strict. That is to say, the PCR nozzle is not tight enough, and the cover of the PCR tube cannot be completely sealed. In this way, it's no use trying to cover it up. There are two ways to solve the evaporation problem. One is to use an air inlet pipe, which can effectively reduce evaporation, but if your system is too small, such as 5 microliters, evaporation may still be obvious. Another method is to use your original PCR tube, but enlarge the system! For example, you used to make 15 microliters, but now you change it to 30 microliters, which ensures that it can be used even if it evaporates!

There are bubbles in the PCR reaction probably because you didn't centrifuge the PCR tube before putting it into the PCR instrument! Usually we leave for a while and get rid of the bubbles! It is impossible to generate bubbles in PCR reaction, because it is only a simple change of heating and cooling, and it does not reach boiling. If there is a bubble, it depends on how big your system is. If it is too small, it will have an impact. In addition, when setting the PCR program, you can set the system a little bigger. For example, if you make a 30 microliter system, you can set it to 40 microliters.

I hope my answer will satisfy you.