Calculations

The OD value of a sample at a wavelength of 260 nm is defined as the extinction that occurs in measuring absorption of the sample in 1 ml aqueous solution in a 1 cm cuvette at the respective wavelength. In practice, OD values are often outside of the linear measurement range of an UV spectrometer; therefore diluted samples have to be quantified and to be extrapolated to the total OD value.

Example:

The dried oligonucleotide is dissolved in 400 µl of sterile water. From this stock solution an aliquot of 10 µl is taken and filled up with sterile water to 1 ml. Now the extinction value of this diluted solution is measured in a photometer in a 1 cm quartz cuvette at a wavelength of 260 nm. The obtained extinction (e.g. 0,25) is multiplied with the aliquotation factor (400 µl/ 10 µl = 40). The result is the OD value (10 in our example).
 

According to the law of Lambert and Beer  (E = Epsilon Ɛ * C * d) one can convert from the extinction E (OD value) to the concentration C and therefore to the amount of substance of  the oligonucleotide. Strictly speaking, the extinction coefficient Epsilon Ɛ is different for each oligonucleotide sequence and would have to be ascertained empirically. In a good approximation it corresponds to the sum of the extinction coefficients of the individual nucleotides and the modifications in the sequence.
Using the oligo´s sequence data and the OD value one can calculate the amount of material as follows: 

E(260nm):  OD-value n [nmol]: Amount in nmol A,G,C,T:  Number of the respective bases in the oligonucleotide Epsilon Ɛ:  Extinction coefficient of attached modifications

For mixed sequences the following values for the amount of material (nmol) result depending on oligo length:

Amount of oligonucleotide in µg

 

Using the amount of substance in nmol and the oligo's molecular weight, one can calculate the amount of oligonucleotide in µg:

n [µg]: Amount in µg n [nmol]: Amount in nmol MW: Molecular mass

Also,  for a mixed 20mer oligonucleotide roughly estimates: 1 OD approximates 30 µg DNA oligonucleotide

 

c [ pmol / µl ] = c [ nmol / ml ] = c [ µmol / l ] = c [ µM ]
 

Example: 248 pmol/µl = 248 nmol/ml = 248 µmol/l = 248 µM
 

The molecular weight of an oligonucleotide is calculated from the number of individual nucleotides in the oligonucleotide and from possible modifications of the oligonucleotide:
 

a) Wallace Rule (2 + 4 Rule)¹

This rule which is valid for very short oligonucleotides (up to about 15 bases) assumes a contribution of 2 °C for each AT-pair and of 4°C for each GC-pair to the melting temperature of a double strand:

b) Calculation by GC-content

The following formula based on Howly et al.^2,3 takes mostly into account the GC content of the oligo and is valid for long oligonucleotides:

with:    
%GC = Percentage of GC-pairs
c(M+) = Concentration of mono-valent cations
n = Number of nucleotides
%F = Percentage of formamide in the buffer
D = Percentage of mismatches

c) Nearest Neighbor Method

The so-called Nearest-Neighbor-Method also takes into account sequence-dependent stacking effects for Tm calculation and is based on thermodynamic properties of neighbouring nucleotide pairs. This method is reliable for oligonucleotides of medium length (20 – 60 bases).

Example: 5'-GTC GAA CCG GAA ACC ACC CCT-3'
 

dH = -6.5 –5.6 –11.9 –5.6 –9.1 –6.5 –11.0 –11.9 –11.0 –5.6 –9.1 –9.1 – 6.5 –11.0 -5.8 -6.5 –11.0 –11.0 –11.0 –7.8 = -173.5

dS = -17.3 –13.5 –27.8 –13.5 –24.0 –17.3 –26.6 –27.8 –26.6 –13.5 –24.0 –24.0 –17.3 –26.6 –12.9 –17.3 -26.6 –26.6 –26.6 –20.8 = -430.6

Tm = (1000 x (–173.5))/ ((-10.8 –430.6 + 1.984 x ln (6.25 x 10-11)) – 273.15 + 16.6 log 50x10-3 = 60.7

All methods for the calculation of melting temperatures given above do not take into account modifications of the oligonucleotide (e.g. dyes, linkers).