My real time PCR run for the standard curve is amplifying too early and amplification of all the dilutions starts on the same cycle. I initially thought the problem was the concentrations of the positive control and I further diluted it and there was no change. I then thought it could be contamination, so I opened new kits, cleaned my pipettes with DNA away, exposed the pipettes, tubes and plates to UV light for 1hr. I repeated the run again and I still got the same outcomes. I use the Primerdesign genesig Kit for Toxoplasma gondii (T.gondii) and run on the Applied Biosystems QuantStudio 6 Flex Real-Time PCR System.