what is gain in fluorescence detector

Cat · Post by **Cat** » Tue Oct 09, 2012 12:37 am

what is gain in fluorescence detector

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Post by Cat » Tue Oct 09, 2012 12:37 am

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 · Post by **jonmoulton** » Tue Oct 09, 2012 10:30 pm

Re: what is gain in fluorescence detector

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Post by jonmoulton » Tue Oct 09, 2012 10:30 pm

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 · Post by **Cat** » Thu Oct 11, 2012 1:44 am

Re: what is gain in fluorescence detector

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Post by Cat » Thu Oct 11, 2012 1:44 am

Thanks! I already solved the problem.

bravebeaker · Post by **bravebeaker** » Fri Oct 12, 2012 12:44 am

Re: what is gain in fluorescence detector

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Post by bravebeaker » Fri Oct 12, 2012 12:44 am

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.

AHH, Gravity! Such a cold cruel mistress.

Cat · Post by **Cat** » Fri Oct 12, 2012 12:58 am

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Post by Cat » Fri Oct 12, 2012 12:58 am

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 · Post by **jonmoulton** » Fri Oct 12, 2012 2:40 pm

Re: what is gain in fluorescence detector

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Post by jonmoulton » Fri Oct 12, 2012 2:40 pm

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

Cat · Post by **Cat** » Fri Oct 12, 2012 10:46 pm

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Post by Cat » Fri Oct 12, 2012 10:46 pm

Yes. It did.