Hi, I am using sybr green reagents to run my real time pcr. I used new filter tips and wipe all the pipettes with RNAsy water before use. The kit and primers are new, and I am using the supplied water.

I performed 40 cycles reaction. The house keeping gene; beta actin first show up after 15 cycle of the reactions and the gene of interest show up after 20 cycles. At about 30 cycles, the no template control; NTC of the beta actin gene seems to be expressing some specific genes. Some of the NTC of the gene of interest is showing up at about 35 cycles as well.

I did not add anything in the NTC. The NTC only contains the primers, water and sybr green reagent.

When checked under the melt curve, the peak of the beta actin NTC that I get is exactly the same peak as the template. It seems like the NTC is expressing the beta actin gene in the late cycle. It is not primer dimer peak. I find it so strange as I did not add in any templates in the control.

There are two peaks in other primer's NTC. But the strange thing is the sample is only showing one peak when check under the melt curve.

I read from the forum that this might be because of the primer dimer formation and the primer dimer acts as the template for amplification. I did try to work out on the primer concentration. I tried a few range of the primer concentration. Apparently, it is still the same. The NTC is still showing up.

I do not have NTC contamination on the normal pcr under 30 cycles. However if I performed the pcr using the same genes for 40 cycles, the NTC will show up. The band of the NTC is exactly the expected band.

I am really exhausted with this NTC showing up after 30 cycles. Tried a few times, moving to another place to do the preparation... All did not work out. Please let me know if anyone can help me! Thank you very much!