Hi does anyone know whether one can set the Applied Biosystems 7500 such that one reads out the signal at a temperature above the Tm of primer dimers such as to avoid unspecific signal. Usually to set a temperature cycle one can only insert two temperatures (in my case 2s at 95°C und 30s at 65°C for 40 cycles). However, when I manually added another step in this cycling stage I had to set it to: 2s at 95°C; 20s at 85°C (where I read out) and 65°C for 30s. So now the first thing is it only accepts times more than 20s to read out at 85°C, which could affect the PCR(?). Secondly the results it showed in the end looked weird, because the programm ended up reading out signal both during heating and cooling, so the values oscillated. Could anyone tell me or send me a screen shot, whether and how one can set the AB 7500 to read out at higher temperatures such as to avoid nonspecific signal from primer dimers?
It would be much appreciated if someone could give me a hint. Many warm wishes and thanks so much in advance for any input!