I'm using no template control (water + reagents) in my reaction in qPCR. IS it normal to have also in the no template control sample fluorescence detected? Normally i have no template plots weaker than templates plots. The detected fluorescenced in no template samples, could be made by self dimer structures? Or from what?
I'm sure i don't have contamination in my products, because already checked.
Thank you!
Laurence Stuart Dawkins-Hall
- You might expect to see minimal fluorescent signal in water only controls but separated from your real samples by > 10 cycles and thus typically above 35 to 40 cycles (ct)
- If you see appreceable fluorescent signal in your water only control then it might be caused by your primers self annealing causing primer dimers
- If you take a look at your melt curves, if you see a second smaller peak in your real samples and the same temp peak but stronger in water only then you do indeed have primer dimers
- That being so, you need to re design your primers and make with programs that screen for hair pin loops and heterodimers
- I have attached an SOP that talks about this in detail
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