What effect on quantifying a fluorescein labeled oligonucleotide using absorbance at 260nm?
The extinction coefficient of DNA at 260nm is around 10,000/mole/base or 10/µmole/base. We use fluorescein isothiocyanate (Isomer 1) in the preparation of our fluorescein phosphoramidite (10-1963). The extinction coefficient of fluorescein (measured as FITC) at 260nm is around 13,700/mole or 13.7/µmole. The fluorescein contribution to the absorbance of a fluorescein labelled oligonucleotide at 260nm is, therefore, about the same as 1 base or about 5% of a 20mer. This may be neglected or corrected for in the determination of the amount of labelled oligonucleotide from an A260 measurement. Note: dA=15.4, dC=7.4, dG=11.5, T=8.7 Fl-labeled ligos: 480nm excitation, 520nm emmission. The extinction coefficient for fluorescein-5-isothiocyanate at 495 nm is 76,000 L/mole-cm at pH 9. Upon conjugation to protein, and by analogy oligo's, the extinction coefficient is decreased by 10%. This would result in a extinction coefficient of approximately 68,000 L/mole-cm. Additionally the extinction coefficient, and fluorescence emission, is very pH dependent (maximum at pH 9).
REFERENCE(S): M. Powell, Glen Research
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10/23/2013