Is a melt curve analysis necessary when running TaqMan ?

Go to the profile of thelongwindingcode
Mar 13, 2018
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Hi,

Is it necessary to run a melt curve when using TaqMan probes? In theory signal should only be generated when the desired product is produced, and not due to primer dimers etc?

I am using the StepOne thermocycler. The software doesn't allow you to add a melt curve when using the TaqMan assay setting. Would you advise running one separately on the plate after qpcr cycling?

Looking forward to your responses!
LWC

3 Comments

Go to the profile of Roberto Rosati
Roberto Rosati 5 months ago

Hi,
performing a melt curve in a qPCR thermocycler is only possible if you have a means to detect a change in fluorescence - e.g. having an intercalating DNA dye in the PCR mix, such as SYBR green.
Since pre-made hydrolysis probe mixes do not contain such dyes, you can't perform a melting curve analysis with them.
You could, of course, add this kind of component yourself, provided it doesn't occupy the same channel as the reporter dye.

On the theory side, a melt curve is not considered necessary for hydrolysis probes; however, you should calculate your assay's efficiency along the dynamic range you wish to explore, and you should only feel confident to report quantities within this range. If your assay falls short on efficiency, it might be that it's amplifying something else along with the desired PCR product.

Go to the profile of thelongwindingcode
thelongwindingcode 5 months ago

Hi there,

Many thanks for your speedy response, I thought this might be the case!

All the best,
LWC

Go to the profile of Roberto Rosati
Roberto Rosati 5 months ago

You're welcome! Take care : )