I attempted qPCR using a protocol I designed based on several related articles. However, I did not obtain any Ct values and am unsure where the issue might be.
Here are the details of my setup:
Mitochondrial DNA Primers:
Reaction Setup (20 µL):
- qPCR Master Mix (Taqman): 10 µL
- Forward Primer: 0.5 µL (final concentration: 0.5 µM)
- Reverse Primer: 0.5 µL (final concentration: 0.5 µM)
- DNA Template: 1–5 µL (30 ng)
- Nuclease-Free Water: Adjust to 20 µL total volume
qPCR Program:
I suspect there may be an issue with the annealing temperature or primer design. Could anyone help identify potential mistakes in my setup or provide troubleshooting tips to resolve this issue?
Asif Shahriar
Md. Mahmudul Hasan
Thank you so much. No, I did not use it. It was for checking CT values, so I used a 96-well plate, the same as for RT-PCR (I mean for mRNA expression). However, since my sample was DNA, I did not need to perform it in a thermocycler, as far as I know. I did not see any option for gradient PCR, but I will have to double-check the machine. And one more thing: if my B-globin gene has an annealing temperature of 55°C, will the use of 52°C for this cause a problem?
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