Calculations
Extinction Coefficient of an oligonucleotide
The extinction coefficient at 260 nm (e260) is a unique physical property of each
oligonucleotide. It is defined as the absorbance at 260 nm of a 1 M aqueous solution
measured at 20 °C in an optical cell with 1 cm pathway (LambertBeer’s law).
Purinic bases show a higher absorption (OD260) than pyrimidinic bases. Interactions
between neighbouring bases, as well as modifications that absorb at 260nm, also
influence optical absorbance. As a consequence, the extinction coefficient strongly
depends on oligonucleotide sequence and composition.
The extinction coefficient of an oligonucleotide can be approximately (error up
to 20 %) calculated using the following formula:
ε_{260}(mM1*cm1)=(15.4*n_{A})+(7.5*n_{C})+(11.7*n_{G})+(9.2*n_{T})
where 15.4, 7.5, 11.7 and 9.2 are the monomer extinction coefficients in mM1 cm1
measured at 260 nm for d_{A}, d_{C}, d_{G} and d_{T}.
Molar amount (N) of an oligo if OD is known
N [nmol] = OD x 100 / 1.54 x n_{A} + 0.75 x n_{C} + 1.17 x n_{G} + 0.92 x n_{T}
n = number of nucleosides of the type X
Example:
Sequence: GAA ATG AGT GCT CAT CAC TAC TTC CGC (27mer)
n_{A}=7; n_{C}=8; n_{G}=5; n_{T}=7 N [nmol] = 12
x 100 / 1.54 x 7 + 0.75 x 8 + 1.17 x 5 + 0.92 x 7= 41.3
Mass amount (m) if the molar amount (N) is known
M[μg] = N x MW / 1000
MW = Molecular weight
Example:
Sequence: GAA ATG AGT GCT CAT CAC TAC TTC CGC (27mer)
n_{A}=7; n_{C}=8; n_{G}=5; n_{T}=7
MW (calculated) = 8219 g/mol
N= 41.3 nmol
M[μg] = 41.3 x 8219 / 1000 = 339
Molecular Weight of an oligo if the sequence is known
MW [g/mol]= 249.23 x n_{A }+ 240.21 x n_{T} + 265.23 x n_{G} + 225.20 x n_{C} + 63.98 x (s1) + 2.02
n = number of nucleosides of the type X
s = number of all nucleosides per sequence
Example:
Sequence: GAA ATG AGT GCT CAT CAC TAC TTC CGC (27mer)
n_{A}=7; n_{C}=8; n_{G}=5; n_{T}=7
MW [g/mol]= 249.23 x 7 + 240.21 x 7 + 265.23 x 5 + 225.20 x 8+ 63.98 x (s1) + 2.02
= 8219.33
T_{M} (melting temperature of an Oligo) if the sequence is known.
The melting temperature TM, characterises the stability of the DNA hybrid formed
between an oligonucleotide and its complementary strand. At TM 50% a given oligonucleotide
can hybridised to its complementary strand.
For your convenience, please use our online calculation tool MOPS or apply the formula
below:
Example:
Sequence: GAA ATG AGT GCT CAT CAC TAC TTC CGC (27mer)
n_{A}=7; n_{C}=8; n_{G}=5; n_{T}=7 s= 27

Sequences with 15 or less bases
T_{M} [°C] = 2(7 + 7) + 4(5 + 8) = 80.0

Sequences with more than 15 bases
T_{M} [°C] = 69.3 + [41(5 + 8) / 27] – (650 / 27) = 69.3 + 19.7 – 24.0 = 65.0
Anneling Temperate (T_{A})

PCR
T_{A} [°C] = [(T_{M}1 + T_{M}2) / 2] – 3

Sequencing
T_{A} [°C] = T_{M} + 3
[
Top of Page]