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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)
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:
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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