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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 (Lambert-Beer’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:

  ε260(mM-1*cm-1)=(15.4*nA)+(7.5*nC)+(11.7*nG)+(9.2*nT)  

where 15.4, 7.5, 11.7 and 9.2 are the monomer extinction coefficients in mM-1 cm-1 measured at 260 nm for dA, dC, dG and dT.


Molar amount (N) of an oligo if OD is known

  N [nmol] = OD x 100 / 1.54 x nA + 0.75 x nC + 1.17 x nG + 0.92 x nT  

n = number of nucleosides of the type X

Example:
Sequence: GAA ATG AGT GCT CAT CAC TAC TTC CGC (27mer)
nA=7; nC=8; nG=5; nT=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)
nA=7; nC=8; nG=5; nT=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 nA + 240.21 x nT + 265.23 x nG + 225.20 x nC + 63.98 x (s-1) + 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)
nA=7; nC=8; nG=5; nT=7
MW [g/mol]= 249.23 x 7 + 240.21 x 7 + 265.23 x 5 + 225.20 x 8+ 63.98 x (s-1) + 2.02 = 8219.33

TM (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:

  • Sequences with 15 or less bases
  •   TM [°C] = 2(nA + nT) + 4(nG + nC

  • Sequences with more than 15 bases
  •   TM [°C] = 69.3 + [41(nG + nC) / s – (650 / s)] 

    n = number of nucleosides of type X
    s = number of all nucleosides per sequence

Example:
Sequence: GAA ATG AGT GCT CAT CAC TAC TTC CGC (27mer)
nA=7; nC=8; nG=5; nT=7 s= 27

  • Sequences with 15 or less bases
  • TM [°C] = 2(7 + 7) + 4(5 + 8) = 80.0 
  • Sequences with more than 15 bases
  • TM [°C] = 69.3 + [41(5 + 8) / 27] – (650 / 27) = 69.3 + 19.7 – 24.0 = 65.0

Anneling Temperate (TA)

  • PCR
  •   TA [°C] = [(TM1 + TM2) / 2] – 3 
  • Sequencing
  •   TA [°C] = TM + 3 


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