The selection criteria for the optimum renaturation temperature should be the intermediate temperature of the amplifiable range.
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PCR Beginner's Guide: Selection criteria for optimal re-temperature
When using the temperature gradient method to find the optimal re-temperature, the novice tends to choose the most temperature conditions for the amplification product, but the amplification effect is not ideal after the non-gradient amplification. The main reason for this phenomenon is that the selection criteria for the optimum re-temperature are not correct. I have done thousands of gene amplifications more than 10 years ago. Through temperature gradient experiments, the temperature range that can be amplified is generally 3-30 degrees. The criteria chosen should be the intermediate temperature of the amplifiable range.
because:
1. There are many factors affecting the amount of amplified products, some of which are random. It can be seen from the irregularity of the platform period of quantitative PCR.
2. The temperature and temperature curves of different instruments and different locations are different. According to the factory standard of the instrument manufacturer, the accuracy is also +-0.3 degrees and the uniformity is +-0.3 degrees.
3. The temperature accuracy of the PCR instrument will be worse when it is subjected to gradient amplification.
Selecting the intermediate temperature eliminates the effects of instrument temperature differences.
In addition, the selection of the amount of the template when performing the temperature gradient is also very important. 
PCR Beginner's Guide: Selection criteria for optimal re-temperature
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