When I perform qPCR using SYBR green, I get amplification in all extracted samples. Because it was doubtful I extracted normal human serum and also water and all those were providing signals. I used the primer blast to see whether the primer was non-specific, but those were ok! However, my NTC was not having signals. have added DNA free water as NTC. But when the water is extracted there were signals. I refused the primer concentration by fourfold with the same results. I have attached here the results.
In case of your negative control, all your Ct values are generally greater than 35, which are nearly insignificant or near to undetermined values.However it could be minimised if you use freshly extracted autoclaved milliQ water, because this type of situation occurs when there is cross contamination.
If it appears late cycle (35+) , it should not be an issue. Especially given that your positive controls and samples appear (~cycle 17).
I agree with previous answers. Ct values greater than 35 are ok for negative control (can be ignored), especially when Ct values of samples are lower than 20.
I also involved in above q PCR, Can somebody explain ,, can we ignore Ct values greater than 35 when they have a melt temperature
If your a-specific product in your control is coming up much later, e.g. 5 or more cycles, than the signal in your sample, the amount that the a-specific reaction will contribute to your signal is only about 3%... thus you might be able to ignore.
Just one additional note. I never use MilliQ water for qPCR, I found that it gave too many problems (not clean enough). I'd recomend commercially purchased ultrapure water
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