I want to evaluate the specificity and development of secondaries structures of my primers and probes against dengue virus (I designed them), I don't know what the usually temperatures for this experiment, i gonna use the FAST 7500 thermocycler by applied biosystems.
any suggestions, I'll be very grateful.
The melting curve is added as a default curve if you choose sybrgreen assay.
If you are using taqman probe assay, I do not think you need a melting curve and it will not show you anything. As the source of fluorescence is the hydrolyzed probes during extension not the DNA.
Usually melt curve is starting with (rapid heating to 94°C to denature the. DNA, followed by cooling to 60°C) with reading fluorescence along the whole time of decreasing temperature.
thank you for your answer,
how many seconds i should put in the software? maybe 60 seconds?
I added this handbook to my previous answer. I think it will help
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