what is gain in fluorescence detector

[Cat]

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.

Thanks.

[jonmoulton]

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.

[Cat]

Thanks! I already solved the problem.

[bravebeaker]

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.

[Cat]

In my case it turned out to be low template concentration. Once I doubled it, everything fell into place.

P.S. I am using Rotor-gene Q for SYBR-Green assays as well.

[jonmoulton]

When you increased the analyte concentration, did the machine calibrate gain before the first cycle?

[Cat]

Yes. It did.