I did a serial dilution of my target starting from 250 ng/ul to 5 dilution steps using 10 fold dilution.

I see good efficiency fro 25 ng ul onwards but I donot see any amplification for 250 ng/ul. Rather the ct value appeared to be 22 but for other steps it was 12, 15 and 18 and so on.

Not sure what happened here. My suspicion is on the inhibitors which got diluted in the dilution that made the reaction work for lower concentration.

I am doing real time PCR using sybr green dye I.