If you used the same volume of each substance at the same concentration, then you have the same number of molecules of each.

A difference in fluorescence intensity of two identical fluorophores at the same concentration and in the same solvent would be surprising. I'm not sure what ssDNA-FQ is, but I assume from your question that it must contain a covalently attached 6-FAM fluorophore, so you expect it to have the same quantum yield as free 6-FAM. This may not be the case, however, because in one case the fluorophore is covalently attached to a DNA molecule, and in the other it isn't. In certain solvent conditions, this could lead to a greater quantum yield for the covalently attached fluorophore than the free one. I think this might be especially true if the pH is acidic.

Also, the way you obtain the concentration of 100 uM concentration of ssDNA-FQ and the way you obtain the concentration of 100 uM of 6-FAM may result in some difference in concentration, denpend the extinction coefficients you used. Was there a significant difference in fluorescence in your case?

Srisruthi

The extiction coefficients (Abs) can be different when Fam attaching to different molecules. The concentration of ssDNA-FQ (Fam lablled ssDNA, right?) can be determined by abs@260(DNA) or by abs@498nm(Fam), this may result difference in concentrtion.

The Fam you used may be only 80-90% pure, with some no fluorescent impurity in.

For concentration of dye, extiction coefficient is used to dertimined the concentration.