Determining oligonucleotide concentration

Introduction

Oligonucleotide concentration can be determined accurately by measuring UV absorbance at 260 nm with a spectrophotometer. According to the Beer–Lambert law:

A = ε × C × l

where A is absorbance, ε is the molar extinction coefficient (M⁻¹·cm⁻¹), C is concentration (M), and l is optical path length (cm). Absorbance is measured at a defined wavelength, temperature and buffer composition, all of which can affect ε.

Calculation

DNA and RNA bases absorb most strongly near 260 nm. Purines (adenine and guanine) have higher molar extinction coefficients than pyrimidines (cytidine, thymidine and uracil), so oligonucleotides with different compositions can have different concentrations at the same measured absorbance.

Base-stacking interactions also affect the extinction coefficient, so the sequence itself must be taken into account. SYNTOL uses a calculation program that considers both composition and sequence.

Pairwise molar extinction coefficients used in the source article are:

Once absorbance A and ε are known, molar concentration is calculated by the Beer–Lambert equation. To express concentration in µg/µL, calculate the oligonucleotide molecular weight from its composition and use C(µg/µL) = C(M) × MW.

Conclusion

The oligonucleotide passport supplied with each order states concentration in µM and in µg/µL.