7_Quantitative PCR -- the melting curve

[@mif3894]

Thank you Mr Dobblestein, the series of your videos has been very helpful.

[@mahmoodpoorjam4639]

You taught melting curve completely and this video was helpfully. Thank you so much.

[@MT-we3fw]

Thank you very much for explaining PCR so well. You are the best teacher!!!

[@MDobbelstein]

Dear Rizzolb,
it doesn't really make much sense to do a melting curve when using taqman. For taqman, the fluorescence signal is based on a the cleavage of a fluorescently labelled oligonucleotide (through exonuclease activity of Taq polymerase). Therefore, the signal will not change if you simply change the temperature without polymerization going on. Hence, taking a melting curve does not make sense in the case of TaqMan PCR.
Best regards, M. Dobbelstein

[@asyifahtheaiskey923]

Thank you Matthias, this video has been of great help! Worth every minute, very interactive and I've now managed to master the key things i need to know when i use rt-qPCR to analyze the relative gene expression of my target genes! (:

[@glen922]

A very nice series, carefully and clearly explained. Thank you very much.

[@liliiademianenko6205]

This course is amazing!

[@nadiak4805]

Thank you so much! This series on PCR has been extremely helpful :)

[@tissarahim4005]

thanks very much for these videos. they are very helpful. I am just curious about the threshold-shouldn't the cycle threshold be taken at the exponential phase of the curve instead of at the beginning of the curve? thanks

[@MDobbelstein]

The first derivative is a mathematical term for the slope of a curve. You may recall from your math classes that the function f(x) has the first derivative f'(x). In the case here, T is displayed on the x axis, RFU is displayed on the y axix, and the slope of the resulting curve is then given by the first derivative, you could call it RFU'(T) or d(RFU)/dT

[@auo2007]

Dear Matthias, I am clinician and now doing Research I am very impressed because you are so clear in describing the melting curve and peaks how they originating from. I just curious to know how about the suspect of mutation, is there anything we could see from melting peaks or curves? I mean only suspect.... Since I did also the melting peaks they are 2 peaks, and when I google there is some explanation from "wikipedia" that the probability is also mutation... How about that?

[@laure189]

Fantastic series of lectures, and extremely helpful! Thank you so much!!

[@kavyavinayan7902]

Vey well-explained videos. Thank you so much. I have a doubt, why the melting temperature always should be 85? Does it depend on the gene of interest that we are looking at?

[@medizinlehre341]

You are right, the melting temperature depends on the PCR product, mostly its length and its GC content. Thank you for your encouraging comment.

[@MDobbelstein]

The first derivative has its maximum (or minimum) at the inflexion points. The second derivative is zero at the inflexion points. For our purpuses, it is easiest to calculate the first derivative and determine its minimum (that is, the maximum of its negative, -d(RFU)/dT.

[@Ahmad26a]

Thanks a lot for these helpful videos .. I am doing a qPCR on miRNAs, could I use some miRNAs as a reference for my assay?

[@rizzolb]

Very good explanations! Helped me a lot! Thank you!

[@rizzolb]

I was thinking about if we use taq-man how the melting curve will look like?

[@perunica1010]

Thank you so much for this series!

[@weronikag7660]

very helpful thank you!

[@somayyehsedaghatjoo4355]

Dear Mr.Dobbelstein
Thanks for your nice clips.But I do not understand what do you mean by"first derivative?"where does it comes from?
Best Regards

[@aheebru77]

lots of Thanks Dr

[@MDobbelstein]

Then it's definitely not the desired PCR product. Short primer dimers could be a source for this.

[@frangallo]

isnt it the second derivative the one that gives you inflexion points?

[@Polarcupcheck]

Very good.

[@feetz88]

Danke schön!!!! :D