Hello everyone!
I ran an experiment to detect L. monocytogenes in chicken sample.
Based from this curve, can you find the problem?
by the way,, my melting curve (curve was not included in the document) got multiple peaks & I was unable to get the standard curve. R^2= 0.3 something
With just the information provided, and the amplification curves, I would say your samples are likely overloaded, those Ct's are really early.
With just the information provided, and the amplification curves, I would say your samples are likely overloaded, those Ct's are really early.
I agree with Craig. it should run internal positive control andnegativw control
Can you tell us the RNA concentration you use for reverse transcription? How much cDNA do you use for qPCR? How much Primer do you use, 1 µL of 10 µM? What about your negative control, do you see peaks in the melting curve and amplification?
Hello Dira, if you are performing the standard curve qPCR analysis, your first task is to establish a reliable standard curve, only then you can start testing your samples. The method is well described in the article (the link below). I am sure you will be able to adjust it to your sample type. Good luck!
Article Detection and Quantification of Listeria monocytogenes in Re...
I agree with Craig. You have a high copy number of template in your sample giving you a very low Ct value. Try diluting the sample and re-running.
You should probably run a standard with different template concentrations for a known gene to optimize the amount of DNA template to be used. Also include a negative control, so that you can see if your multiple peaks in the melting curve indicate primer dimers or non-specific amplification.
Estimated your DNA quality and quantity first. As per suggestion by Irina to check detection limit of primers using standard curve. than proceed for further. If Melting Curve showing multiple peak that's means lot of background noise due to excess of master mix or nonspecific amplification. Check your set of primers specificity also using Bioinformatics' tool too.
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