Hi,
I've recently used a pure spectra plate from Thermo to calibrate my QPCR. Upon review of the raw data, I noticed that the SYBR spectra had 2 peaks (well a peak and a shoulder peak). Is this typical? The 'shoulder' peak falls in the range of another dye so would this affect quantification of other dyes if multiplexed?
Thanks in advance
David
ps - picture of spectra shown
Hi David,
Based on the available data on thermofisher website, the emission peak of SYBR-GREEN I is asymmetric, but doesn't display any shoulder as you show.
If this "shoulder" peak overlaps the emission of another dye, it will cause bias in the quantitation of this other dye. It is possible to do a correction by substracting the amount of "green" in the "red" channel. For example Red_corrected = Red_Raw - X%Green_Raw.
Are you sure you are measuring the emission of SYBR-GREEN alone or both fluorophores in your spectra plate ?
You should also show on the x axis the wavelengths to have a better idea of what we are looking at.
Cheers
Oliv
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