Hi!
we are seeing high fluorescence amplitude (4000) in the NTC, similar to the tested samples. We know we do not have contamination in the Mastermix because we have opened a new vial and we still see the same. We are not sure whether we have to high signal in the NTC or if we have to low signal in the samples. We are running a cDNA amplification of 40 cycles, stained with EvaGreen. Does anyone have any suggestions? Thanks
Depending on the amplification curve profile I would suggest to add a positive control to be sure that you obtain the maximum fluorescence. It will respond to your first question.
Finally, especially in green staining, a high number of cycle could generate false amplification. Maybe it is responsible of your signal in NTC ?
I hope it will help.
Maxime
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