I’ll be doing qPCR using probe based primers. The primers and probes are premixed. I’ve heard that before optimising qPCR, a thermal gradient PCR has to be done. Could I do a thermal gradient PCR using my premixed primers and probes? If so, what are the parameters?
Harpreet Kaur Ranjit Singh You can indeed do a thermal gradient qPCR run with your premixed primers/probes. I included a file (from a commercial distributor) that gives an example how to optimize your qPCR. Best of luck with your experiment.
Also you might check out this link underneath as it gives you an idea on how to proceed with primer optimization.
https://www.sigmaaldrich.com/technical-documents/protocols/biology/primer-optimization-using-temperature-gradient.html
In qPCR, you optimize for rapid amplification, meaning early ct. The regular PCR gradient doesn't tell you what the ct was. So I'm not entirely sure what you would learn from that.
You'll likely see that product is produced across a broad range of conditions, but you won't know which one is optimal for qPCR.
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