Is it possible a DNA with an absorbance ratio A260/A230 = 2.96, for example, be influencing the low fluorescence in the Real-Time PCR? I was trying to optimize a real time PCR method to amplify a specific segment and for this purpose, I was using primers which were designed for a conventional PCR and the fragment lenght is 200bp. Could be this the problem which is causing the low fluorescence?
I've already used different SybrGreen Master Mixes...