when I´m running qPCR (standard is plasmid DNA with insert of our interest) with 5 standards (10-fold dilutions) I still have PCR efficiency around 160% or more (with Sybr and Probe as well)-slope around -2,55.
Moreover I tried to delete some dilutions to overcome this but result is even worse. I didn´t use touchdown (what I´ve heard can slightly increase efficiency) and I know that only 1 product is amplified (according gel elfo and HRM).
I´ve tried different reactions (completely different inserts in the same plasmid) still having this too high efficiency and delta Ct is still around 2-2.5.
Any explanation or suggestions?