when I use SYBR Green II nucleic acid gel stain (1:10000 in TBE buffer) with mIRNA, why fluorescence decrease with increasing the concentration of mIRNA? I tried many times but same. I select the excitation is 579 nm and emission is 500-600 nm.
The answer I could think of is that since fluorescence occurs when we excite the nucleic acid gel stain, the more concentration of it you have, the more molecules you need to excite, the more energy needed. Thus it decreases fluorescence...or at least slows it down. Perhaps try to increase the excitation up to 585-590nm?
Possibly if the concentration is already high then the emitted light could be re absorbed by non excited dna molecules attenuating the emission at shorter wavelengths. If the sample is very concentrated possibly the excitation light may all be absorbed and not penetrate the full sample leading to a plateau in the emitted light
Clearly since the nucleic acid gel stain is a buffer that has the capacity with the ration 1:10000, therefore when it will increase its concentration therefore the fluorescence will also decrease 10000 times.
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