Some experimenters often overlook a key thing when performing fluorescent quantitative PCR experiments: Annealing temperature of the primers of the gene to be tested.
First of all, when we want to measure the expression of a certain gene, we need to design primers. At this time, we will encounter two situations:
- This gene is known and has been studied by predecessors;
- This gene is unknown and you need to explore it yourself.
So, if our gene is unknown, you need to explore it yourself. But how to explore it?
Firstly, we need to select a proper temperature:
→ There will be an instruction sheet when the synthesized primers are sent over. There will be the Tm value of the upstream and downstream primers, which is the melting temperature of the primers. Then you may get the result you want!
Attention: This melting temperature and annealing temperature are not the same things! Generally, the annealing temperature is about 5°C lower than the melting temperature.
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