Dear friends,
I need to know about melting curve in qPCR (Syber Green) cycle conditions.
In the following paper, the context indicates melting curve from 72 C to 95 C for 15 min.
Article Reagent and laboratory contamination can critically impact s...
My question is which part of qPCR steps is (denaturation, annealing or extension) melting curve or melting curve condition placed in? In other words, where does among the three main PCR steps the melting curve procedure happen in? Or is the melting curve a seperated step from the three main steps?
Thanks a million
Mehrdad
Hi,
As far as I know, the melting curve condition was conducted after the three main steps (post-amplification). Post-amplification melting-curve analysis is a simple, straightforward way to check real-time PCR reactions for primer-dimer artifacts and to ensure reaction specificity.
Best regards
Hi,
Melting curve is done post pcr to check the primer dimer or specific product formation.
Temparature setpoint is decided based on the product Tm. for pcr products you can run 60 to 95 with 1% temparature ramp rate. (The ramp rate may vary in different qPCR instruments).
Hope this helps
https://youtu.be/m_IO5cjJFQ4
Ajith Kumar sorry, may I ask, if the Tm peak is below 80℃，does this mean the peak is not the target I am amplifying? mine is 78.2℃. thank you:)
- Badges
- Science topic
- Similar topics
- Engineering
- Materials Engineering
- Annealing