Property Calculation Tools

Property Calculations

Molecular Properties
Design Tools

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Molecular Weight

The molecular weight of a substance is the weight of one mole of a that substance. To calculate the molecular weight of an oligonucleotide, the molecular weights of the individual nucleotides present in the oligo must first be summed. Then the weight must be corrected by subtracting 62.97, which is equal to two oxygen atoms and a phosphorus atom, to account for the 5’-terminal OH group, and by adding 1.008, which is equal to one hydrogen atom, to account for the 3’-terminal OH group.

The nucleotide unit:

For DNA: R = H; “Base” can be Adenine, Cytosine, Guanine, or Thymine.

For RNA: R = OH; “Base” can be Adenine, Cytosine, Guanine, or Uracil.

DNA: MW = (#dA x 314.21) + (#dC x 289.18) + (#dG x 329.21) + (#dT x 304.19) – 62.97 + 1.008

RNA: MW = (#A x 330.21) + (#C x 305.18) + (#G x 345.21) + (#U x 306.17) – 62.97 + 1.008

To calculate the molecular weight of Oligo Potassium Salts, the molecular weight of the normal oligonucleotide must first be calculated (see above). This weight is then corrected by adding 39.1, which is equal to one potassium atom, and subtracting 1.008, which is equal to one hydrogen atom. This correction accounts for replacing all the acidic protons on the backbone with potassium ions.

K-Salt: MW = MW(normal) + (# nucleotides -1) x (39.1 – 1.008)

Melting Temperature

The melting temperature of an oligonucleotide is dependent upon a number of factors including the length and G+C content of the sequence as well as the type and concentrations of cations present, particularly the sodium ion, Na⁺. A variety of formulas have been used for predicting melting temperatures. The following formula is recommended for oligonucleotides ranging in length from 20 to 100 residues and sodium concentrations ([Na⁺]) ranging from 0.01 M to 1.0 M.

Extinction Coefficient

e₂₆₀, the extinction coefficient at 260 nm, is a constant that indicates the extent to which a given DNA or RNA strand in solution absorbs light at 260 nm.
In general, the calculation of the extinction coefficient of an oligonucleotide of length N can be given by the expression:

e_N is the “nearest-neighbor” extinction coefficient of neighboring nucleotidesiandi + 1.
Nearest-neighbor extinction coefficients at 260 nm:

	i = C	i = G	i = A	i = U	i = dC	i = dG	i = dA	i = dT
i + 1 = C	14200	17800	21000	16200	14400	17900	21050	15200
i + 1 = G	17400	21600	25200	21200	17500	21600	25200	20000
i + 1 = A	21000	25000	27400	24000	21100	25000	27400	22800
i + 1 = U	17200	20000	24600	19600	17200	20000	24600	18200
i + 1 = dC	14400	17900	21050	16200	14600	18000	21100	15200
i + 1 = dG	175000	21600	25200	21200	17600	21600	25200	20000
i + 1 = dA	21100	25000	27400	24000	21200	25000	27400	22800
i + 1 = dT	16200	19000	23400	18200	16200	19000	23400	16800

e_I is the “individual” extinction coefficient for nucleotidei.
Individual extinction coefficients at 260 nm:

i = C	i = G	i = A	i = U	i = dC	i = dG	i = dA	i = dT
7200	11500	15400	9900	7400	11500	15400	8700

Molarity

Molarity is a measurement of concentration. It is equivalent to moles (mol) of solute per liter (L) of solution as expressed in the equation below.

Absorbance

The concentration of oligonucleotide in a sample is measured by its absorbance at 260 nm according to the Beer-Lambert law.

where A is the absorbance (optical density or OD), ε is the molar extinction coefficient, c is the concentration, and l is the pathlength.

Annealing Temperature

To calculate the annealing temperature of oligonucleotides, first calculate the melting temperature (see above), then subtract approximately 5–10 degrees. If the two oligonucleotides have different melting temperatures do NOT average the numbers. Use the lower number so that both of the oligonucleotides can anneal. For 10mers, Eurofins Genomics recommends that PCR be done at 32–33 °C in order to maximize repeatability.

Picomoles

Picomole is a measurement of amount. The following equation can be used to calculate picomoles from the optical density (OD) and extinction coefficient (e₂₆₀) of the oligonucleotide.

Micrograms

Microgram is a measurement of weight. The following equation can be used to calculate micrograms from the picomoles (pmol) and molecular weight (MW) of the oligonucleotide.

Note: for an oligonucleotide 1 OD₂₆₀ = 33 µg.