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How to measure OD’s of Oligonucleotides

In the biochemical lab, there is the need for measuring nucleic acids, DNA, RNA, LNA's and modifications thereof (including fluorescent attachments, bio-conjugates and other modifications)

Definition: One absorbance unit is defined as the amount of nucleic acid that will produce a reading of one, when measuring a one mL volume in a one cm quartz cuvette at 260nm wavelength; in fact this is derived from the Beer-Lambert equation.

A = ebc

• A is absorbance (no units, since A = -log₁₀ P₀ / P )

• e is the molar absorptivity (also known as the molar extinction coefficient) with units of L mol^-1 cm^-1

• b is the distance the light travels through the sample contained in the cuvette. We will express this measurement in centimeters.

• c is the molarity of the mixture, expressed in L mol^-1.

The individual extinction coefficients for each four DNA bases are:

• A has absorbance maximum at 259nm, extinction coefficient = 15.4 × 10³ M^-1 cm^-1 (pH 7.0).

• C has absorbance maximum at 271nm, extinction coefficient = 9.0 × 10³ M^-1 cm^-1 (pH 7.0).

• G has absorbance maximum at 253nm, extinction coefficient = 13.7 × 10³ M^-1 cm^-1 (pH 7.0).

• T has absorbance maximum at 267nm, extinction coefficient = 9.6 × 10³ M^-1 cm^-1 (pH 7.0).

From these values we can observe where we derive the value of 10 × 10³ M^-1 cm^-1, which is the average extinction coefficient of the 4 nucleotides.

Practical formula: A very simple and useful formula to obtain molarity is the following:

micromoles of an oligo = 1 / (N × 10)

Where

N = oligo length
10 = the average extinction coefficient of all 4 nucleotides

Example: How many micromoles are there in 1 OD of a 20mer oligo?

# of µmols = 1 / (20 × 10) = 0.005 = 5 nanomoles

Useful DNA data approximations:

• 1 A₂₆₀ of long SSDNA = 37 µgs
• 1 A₂₆₀ of oligo ss DNA = 33 µgs
• 1 A₂₆₀ of oligo ssRNA = 33µgs
• 1 A₂₆₀ of dsDNA = 50 µgs
• 1 A₂₆₀ of dsRNA = 40 µgs

Note: the quenching effect upon duplex formation, the absorbance is not the exact addition of the individual strands, rather is about 1.5 times (not twice as one would expect).

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