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 260nmm wavelength; in fact this is derived from the Beer-Lambda equation.
A = ebc
*
A is absorbance (no units, since A = log10 P0 / P ) *
e is the molar absorptivity with units of L mol-1 cm-1 *
b is the path length of the sample - that is, the path length of the cuvette in which the sample is contained. We will express this measurement in centimeters.
The individual extinction coefficients for each four DNA bases are:
*
A= Absorbance maximum at 259nm, extinction coefficient = 15.4x103 M-1 x cm-1 (pH 7.0).
*
C= Absorbance maximum at 271nm, extinction coefficient = 9.0x103 M-1 x cm-1 (pH 7.0).
*
G= Absorbance maximum at 253nm, extinction coefficient = 13.7x103 M-1 x cm-1 (pH 7.0).
*
T= Absorbance maximum at 267nm, extinction coefficient = 9.6x103 M-1 x cm-1 (pH 7.0).
From these values we can observe where we derive the value 10, which is the average extinction coefficient of the 4 nucleotides.
Practical formula: A very simple and useful formula to obtain molarity is the following:
# of nanmoles of an oligo= 1/Ix10
Where
I = 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 ?mols = 1/20x10 = 0.005 = 5nanomoles
Useful DNA data approximations:
* 1 A260 of long SSDNA= 37 ?gs * 1 A260 of oligo ss DNA= 33 ?gs * 1 A260 of oligo ssRNA=33'gs * 1 A260 of dsDNA=50 ?gs * 1 A260 of dsRNA= 40 ?gs
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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