QUANTIFYING DNA
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The quantity of the oligonucleotide may be expressed in terms of A260 units, nanomoles, or micrograms.
The definitions of these units and conversion factors are listed below:
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A260 unit is that quantity of material which, when dissolved in 1.0
milliliter of water and read in a spectrophotometer at 260 nanometers in a cuvette with a 1 centimeter path, gives an absorbance of 1.0
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An oligonucleotide with an evenly-distributed base composition which has
an absorbance of 1.0 at 260 nanometers has a concentration of approximately 30 microgram/milliliter.
The base composition has a dramatic effect on this approximation, since a sequence of:
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All A's = 20 micrograms/milliliter when A260 = 1.0
All T's = 35 micrograms/ milliliter when A260 = 1.0
All C's = 39 micrograms/ milliliter when A260 = 1.0
All G's = 26 micrograms/ milliliter when A260 = 1.0
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The number of nanomoles of an oligonucleotide is calculated from the measured
A260 value
using the millimolar extinction coefficient (mM E260) of the specific oligonucleotide.
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Nanomoles = 1000(Measured A260 value x dilution factor x total volume)/
(E260 of bases + E260 of modifiers)
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The mM E260 values MIDLAND uses are:
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A = 15.1 | G = 11.7 | T = 8.7 | C = 7.4 | I = 6.8
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For example, a 2.0 milliliter sample with the sequence 5'-ATC GAT CGG ATT-3' has a
measured A260 value of 0.329 when measured at a 1:11 dilution. The total number of
nanomoles in the sample is calculated as follows:
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Nanomoles = 1000 x 0.329 x 11 x 2.0 / 3(15.1) + 3(11.7) + 4(8.7) + 2(7.4) = 55.7 nanomoles
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The number of micrograms of oligonucleotide is calculated using the molecular weight (MW)
and the nanomole value (which was calculated from the A260 measurement).
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The molecular weight of the free acid form of DNA can be calculated as follows:
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Enter the number of A's__________ x 313.22 = __________
Enter the number of C's__________ x 289.18 = __________
Enter the number of G's__________ x 329.22 = __________
Enter the number of T's__________ x 304.21 = __________
Enter the mass contribution of any modifications = __________
Subtract (there is one less phosphate than nucleoside) - 61.98
The molecular weight of the oligonucleotide is = __________
Micrograms (ug) DNA = MW x nanomoles / 1000
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As a general rule of thumb, for an oligonucleotide with
an evenly-distributed base composition with sequence length of 20:
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| 1 A260 unit = 30 micrograms = 5 nanomoles
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