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Real time. No amplification but hight flourescence

real time qPCR

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#1 Anap

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Posted 29 October 2013 - 07:11 AM

Hi,

 

I'm performing real time PCR and in the last 3 trys I have this problem.

 

The flourescence curve start to elevate at cycle 5 for ALL the samples (some of them were differents dilutions). I rich hight levels of fluorecesce, I did not see any product in an agarose gel, and the melting curve is a constant (not change of the fluorescence whit temperature).

 

I can't undertand why I have flourescence whihout a product. Is not a problem of primer dimer cause some of them had been used previously. Is not also the kit, because I check whit three differents brands. I also use two different primer concentration.

 

Please help me! I need those results!



#2 Trof

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Posted 31 October 2013 - 06:44 AM

So you have a SYBR Green assay? 

What is "high levels"? Can you compare the numbers on the raw data graph with some of the previously run samples, the baseline fluorescense and the final one? (not the graph itself, as graphs could be changing y-axis resolution depending on the maximum fluorescence in run).

Your non template control also has a fluorescence? 

How much template do you have in your reaction?

Can you post an image of the melting curve?  (And the raw data graph, while at it).


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