Did a specificity test of newly designed primers. They have been working well, no abnormalities, until tested on human DNA. 2 samples were negative, but 3 were positive, with non-exponential amplification curves.
Does anyone know what might cause this?
Also these positive samples had double-peaked melting curves.
The products have not been analyzed further by gel electrophoresis, as the experiment did not require this. But I am eager to learn, and really wonder what might cause these weird curves..
Hello Kaja,
From the figures that you have attached, I would conclude that the amount of your DNA is not sufficient. To illustrate my point I have added a picture of my DNA serial dilutions experiment, whereby you see a 1000x dilution still gives you a nice S-shape amplification curve and from 100,000 to 1,000,000 dilutions give a linear amplification.
About the double peaks in your peaks in your melting curve, I would think it is caused by primer dimer. I think the first peak is the primer dimer and the second one is the actual PCR product, but due to the low DNA concentration the peak is very low. The second peak is almost at the same place as your positive control (the green peak). You can avoid this by increasing the annealing temperature of your PCR-profile. I would suggest to repeat the experiment in total 3 to 4 more times and each time increase the annealing temperature by 1°C. Please make sure you do your experiment in triplicates.
Furthermore, I see your melting temperature is very high. Actually very close to your denaturation temperature. One way would be to shorten the length of your primers without losing their specificity and make sure your PCR product does not have a big size.
Good luck,
Ali
Hi Keja,
Ali has said it all! Watch out for your melting temperature so as not to denature. Always repeat experiments do not rely on only one set..
Good luck.Iliyas.
I generally agree with Ali and Ilyas. Notice that results coming from two close melting curves cannot be considered as signals.
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