Finding Multiple Melt-Curve Peaks When Using SYBR® Green in Real-Time PCR -- Ask TaqMan®: Ep. 8

[@Ali-fk7cn]

I hope everyone can explain things as you did!! Simple, understandable, brings to mind many informations, thank you

[@thermofisher]

Our pleasure! Thank you for watching.

[@tojama]

I should add that double peaks may also be purely related to the unequal CG distribution within the amplicon (for example, low GC content in the extremes and high in the middle produces two peaks). This does not make your primers bad, but of course will complicate melt curve analysis.

[@pfrtlmpf]

Thanks a lot! This maybe explains finally these annoying shoulders in some of my melting curves. :)

[@bobu5213]

This video was amazing

[@vinodyadav-fi7vd]

first time i felt explanation directly going in my brain

[@DhfdfgbFbdsgfgfh]

Thank you very much..... It is very helpful lecture 🏵️🏵️🏵️

[@bimalcb]

Does a melt curve analysis from microRNAs look different? Especially if the cDNA were prepared by adding a polyA tail(to enrich for microRNAs) to them and hence having a long GC depleted region at the extremes

[@Lineroested]

Thank you very much! That was just the help i needed!

[@pegs19]

Thank you so much for your clear explanation! You made it easy to follow and understand!

[@vebjrnthunes9199]

That is an excellent explanation. I've read that the melting temperature is defined differently in other places. Wikipedia: The temperature at which 50% of DNA is denatured is the melting temperature. Not where all DNA I denaturated.

[@samiraabdi2058]

Hiii, I have a question around these small peaks. Well it is known that in case of any primer dimer or hairpin there will appear a small peak (around 70 degree C), below the trashold line, before the real sample peak(around 80degree C). But, what if this small peak appear after the sample peak (around 90degree C)????

[@thermofisher]

Thanks for your question Samira. The secondary peak that appears after your real peak is some non-specific amplification and that would suggest that some reoptimization may be required. If you have additional questions or would like us to look at your data more closely, please email our technical support team at thermofisher.com/askaquestion. Thanks!

[@samiraabdi2058]

@@thermofisher thank you soo much, I will do so.

[@mehmetyavuz3411]

In order to confirm low level amplification curves obtained by Taqman hydrolysis technology, Can I use this amplified product and current primers only to determine the melting temperature by using Double stranded DNA binding dyes such as SYBR green

[@JatnaRD]

Doug identified as TaqMan :)

[@normapadilla8939]

Good video. Loud and clear. There is a new version of this one, with a lady instead. It is however exactly the same script! identical! if it is not adding anything new...what's the point of the new version ?

[@neuroether]

I am confused. I understand your logic but question if that is the only explanation. If a sequences were subject to computational evaluation of HRM melt curves in a predictive model (iMELT), then the pattern calculated seems nearly identical to the multiple peaks seen in empirically derived HRM using genomic DNA and primers that confine the same stretch of DNA. How is this possible given your explanation that Sybr green derived mulitple peaks are seen from DNA contaminants or primer/dimer intercalations?

[@ozgeberrak1447]

Thank you! :)

[@thermofisher]

You're welcome!

[@tianjiang8861]

I'm using StepOne instrument to perform sybr green based qPCR. However, I got inconsistent results with the same input sample in different trials. For example, I may got a single melting peak at one time, but more than one peak another time using the same input sample. Has anyone ever seen similar problems?

[@tianjiang8861]

***** Tm for the second peak is usually around 65C. It's probably primer dimer, I guess. It looks more like a bump, but the machine determines it's a second peak. The thing is, even in the triplicate for the same sample/primer, one may have a second peak, but one may not. The primers are designed over an exon junction. I didn't use DNase or RNase H. I'm using the comparative CT method. The results seem to be quite inconsistent for different biological replicates.

[@MichaelKoksharov]

You didn't tell when two peaks, for example, are actually ok.

[@ealafabbas9745]

please help me l cant understand relative and absolute quantitation 😢

[@thermofisher]

In relative quantitation, the end result is a relative comparision, such as “gene A is expressed 5 times higher than gene B”. In absolute quantitation, a standard curve is used to determine the exact copies present of the target gene. For more details, please see this comparison table: www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/absolute-vs-relative-quantification-for-qpcr.html

[@nateliechuah1763]

I am performing melt curve analysis using bacterial genomic DNA to determine the G+C content of my bacterial DNA. I am using Applied Biosystem 7500 RT PCR, SyBr Green I, and a detection of fluorescence was set on a ramp from 25-99C. The melting curve is horrible wit a lot of peaks. DNA concentration used is 3microgram per reaction and purity is good. I had ran the DNA samples on agarose gel and only single intact band was observed for all my bacterial DNA samples. Pls advise.

[@lifetechnologiescorp8544]

Hi Natelie. How do the peaks look in the derivative view? What Tms are you getting for the different peaks? If you ran any NTC wells, did those give a peak? If so, then the extra peaks you are seeing are likely from primer dimers. You could try lowering the primer concentration. For SYBR Green, usually around 200-300 nM is best. We hope that helps, and let us know if you have any other questions.

[@nateliechuah1763]

Dear life tech corp. Omg. I just received ur comment which was 7mths ago. I could not get single peak in derivatives view,thus no tm. Ntc no peak. I m not putting any primers. I merely use sybr green to total genomic DNA. It is nt a pcr reaction but directly n only melting curve analysis on da bacterial total genomic DNA. So I m sure no primer dimer prob. I did temperature increament fr 25-99. Sybr signal reduces linearly fr 25-99 n thats why I could not get a peak for tm

[@afaqhameed2843]

Three micrograms is a very huge concentration for melt curve analysis usually it should be 20 to 50ng and the amplicon size is very much important in melt curve analysis