Can anyone explain what GAIN in fluorescence detector actually is? I am running real-time PCR and the signal seems too low for detection if I calibrate gain "before the 1st cycle". If I recalibrate in the middle of the run, I see signal.
P.S. If you do SYBR Green PCR, please, let me know what gain you use.
Gain is the amount of signal amplification. I don’t know why you need to set the gain in the middle of the run. Here’s a guess. Perhaps with no signal, the gain is set low, perhaps to avoid setting the highest possible gain when calibrating against an absent signal then blowing out the detector when a strong signal is initiated with the gain set too high. With some signal, the gain is set so that the signal falls somewhere toward the middle (?) of the range.
care to elaborate how you solved the problem? I used to use Roche light cycler 480 and now I am using RotorQgene and for the RotorQ it optimizes the gain before the run and sometimes during the run. I also found out that the intensity of the fluorescnce depends on how many tubes are in the rotor. I use Syber green by the way.