What causes two peaks in melting curve-Real-Time PCR

Hello everyone. My day was ruined by these two peaks~~~ It's supposed to be one, which represents single product after amplification. Does any one know the reason? I've checked the sequences of both primers. Blast gives single targeted gene sequence. -meflower-

QUOTE(meflower @ Jul 11 2006, 08:33 PM) [snapback]59178[/snapback] Hello everyone. My day was ruined by these two peaks~~~ It's supposed to be one, which represents single product after amplification. Does any one know the reason? I've checked the sequences of both primers. Blast gives single targeted gene sequence. A picture would help...but depending on the location of the the extra peak, it could be primer dimer or gDNA contamination. -medhan1012-

Hello, I also have got problems with my melating curves: The three curves that peak at 83C are my samples, triplicates. The curves that peak at around 50C are my NTC triplicates. What does the shoulder mean in the curves of my samples? If I dilute my cDNA, the second peak gets worse. Has anyone an explanation? Can I use the primers? Many thanks.

-peetyswoo-

QUOTE(peetyswoo @ Jul 12 2006, 08:05 AM) [snapback]59286[/snapback] Hello, I also have got problems with my melating curves: [attachmentid=1308] The three curves that peak at 83C are my samples, triplicates. The curves that peak at around 50C are my NTC triplicates. What does the shoulder mean in the curves of my samples? If I dilute my cDNA, the second peak gets worse. Has anyone an explanation? Can I use the primers? Many thanks. The shoulder often means a smear.You say that if you dilute the cDNA, the other band gets worse. It sounds to me that this is not the most efficient, try some other temperatures. -aspergillie-

Have your thought about of possible alternative splicing fragment of your target gene?

-rye-

Thanks, everybody. I will furthur check it.

-meflower-

QUOTE(meflower @ Jul 12 2006, 09:04 AM) [snapback]59320[/snapback] Thanks, everybody. I will furthur check it. Both these methods will help you answer your melting point queries:1. Run the reactions on an agarose 1000 gel with appropriate DNA ladder and you will see if you are seeing a single band or smearing2. Clone the reaction into TOPO TA vector and sequence inserts to confirm the amplicon is correct. -JPStewart-

if you are ever concerned about gDNA contamination, you can always run a control with gDNA from your cells and see where the peak falls...that way you would know for sure I have found with one of my amplicons that 'shoulders' disappear when I add a bit less primer. I think this would correlate with the shoulders increasing if you add too little cDNA? it is all about the proportion of primer to amplicon and how it affects the efficiency.. good luck

-aimikins-

The second peak is most likely amplification of primer-dimers. This is why you see an increase when you dilute your cDNA and you see a peak in your NTC. The way to avoid this is to ensure you are using a large enough amount of template cDNA.  

-rtilley-

My NTC does not show a graph like my cDNA samples. It does not show a peak.

-peetyswoo-

Dear all, I've had this problem of 2 melt peaks for 2 runs for standard dilution curves in triplicate. Each time the same. The lower concentration dilutions have the lower melt peak (~77) My product is supposed to be ~83. There's no problem for higher concentration dilutions. What's the problem? Do you think I should reduce primer concnetration or do 5X dilution curves instead of 10X? After gel run there were quite obvious primer dimer bands which seem to get brighter as the dilution curve progresses. I use SYBR Green I Master mix from Qiagen, Rotor Gene 3000, cDNA samples (2.2ul), primer conc. of 10pmols (0.5ul each) in a 25ul reaction. I have vortexed and spinned down all my reagents well. My -C also shows the melt peak at 77C but my NTCs have no melt peak. Please help me solve this problem. I don't want to waste more master mixes before I know what's wrong... Thanks.... Chris.

-chris_sylim02-

QUOTE(chris_sylim02 @ Jul 16 2006, 09:18 AM) [snapback]59822[/snapback] Dear all, I've had this problem of 2 melt peaks for 2 runs for standard dilution curves in triplicate. Each time the same. The lower concentration dilutions have the lower melt peak (~77) My product is supposed to be ~83. [attachmentid=1322] There's no problem for higher concentration dilutions. What's the problem? Do you think I should reduce primer concnetration or do 5X dilution curves instead of 10X? After gel run there were quite obvious primer dimer bands which seem to get brighter as the dilution curve progresses. [attachmentid=1323] I use SYBR Green I Master mix from Qiagen, Rotor Gene 3000, cDNA samples (2.2ul), primer conc. of 10pmols (0.5ul each) in a 25ul reaction. I have vortexed and spinned down all my reagents well. My -C also shows the melt peak at 77C but my NTCs have no melt peak. Please help me solve this problem. I don't want to waste more master mixes before I know what's wrong... Thanks.... Chris. I also think it's because of primer dimers. And I think you can see it on the gel (lower band). Try to use less primers and more DNA -dnafactory-

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