The extinction coefficients of the single strands are added together to provide the final data on our Certificates of Analysis. When oligonucleotides are annealed; however, these constants are not entirely additive which can cause confusion when calculating the amount of product present in a sample. Duplex extinction coefficients can be 15-25% lower than expected due to the hyperchromicity of the duplex. Therefore, we provide a calculation on our duplex Certificates of Analysis to help better determine the actual amount of duplex present in a sample:
(Measured ODs/Total ODs of single strands) x Total Extinction Coefficient = Duplex Extinction Coefficent
On the Certificate of Analysis you received with your product, you are supplied with two numbers needed for this calculation: the number of OD260 units of product (the absorbance reading you would obtain from a 1 cm cell path if you dissolved your sample in 1 ml of water, and usually serially diluted for the reading) and its calculated extinction coefficient (ε). The ε is given in units that are readily used for our application, OD260 units per μmole. Divide the number of OD260 units by ε to calculate the number of μmoles directly. For example, if you obtain 60 OD260 units of an oligonucleotide with an ε of 300 OD260units/μmole, you have received 0.2 μmoles of product.
The optical density unit, or more commonly the OD260 unit, is a spectrophotometric measurement of an oligonucleotide. It is a normalized unit of measurement that is defined as the amount of oligonucleotide required to give an absorbance reading of 1.0 at 260 nm in 1.0 mL of solution using a 1 cm light path. Each of the bases in a nucleic acid strand has an absorbance at or near 260 nanometers, due to their conjugated double bond systems. Because the exact base sequence and composition is known, the OD260 unit is a precise method to quantify an oligonucleotide. Utilizing absorbance measurements is the recommended method for quantitating or aliquotting an oligonucleotide.
The molecular weight of your oligonucleotide is provided on the Certificate of Analysis. Multiply that number by the number of μmoles. For example, if the molecular weight of your oligonucleotide is 6950 au, and you have 0.2 μmoles of product, you have 1390 μg or 1.39 mg.
First determine the number of μmoles you have. Next, convert the units of your desired stock solution to M. For example, if you wish to prepare a 10 mM stock solution first convert your units to M by dividing by 1000 (resulting in 0.01 M in this example). Now simply divide the number of μmoles you have by the desired concentration of your final solution in M to determine the μL of buffer you will need to achieve that concentration. For example, if you have 0.2 μmoles of product and you wish to prepare a 10 mM stock solution, divide 10 mM first by 1000 to convert to 0.01 M, and then divide 0.2 μmoles by 0.01 M to obtain the number of μL needed, 20 μL in this case. This is a simplified, working version of the string of mathematical equations that underlie the procedure described. If you require more information, please contact us.
The extinction coefficient, ε, is a physical constant that is a key component of Beer’s Law regarding the relationship between optical absorption and concentration; A = ε [conc]. By knowing the ε of an oligonucleotide one can readily convert the optical absorbance reading into concentration, and then into mass. The ε is based on the actual bending and vibration of the bonds and takes into account the absorbance of the individual bases and the effects of neighboring bases. The ε is derived from the exact nucleotide composition of the oligonucleotide so it is unique to every oligonucleotide sequence. The units for the ε are expressed normally as OD260 units /μmole. TriLink calculates the ε of an oligonucleotide using the nearest neighbor model which is considered to be the most accurate method without actually determining the ε empirically, which requires a great deal of experimental effort.
It is a very accurate and convenient method of quantifying the oligonucleotide because each of the bases has an absorbance at or near 260 nanometers, so it is the average maximum wavelength of long DNA strands. This method has traditionally been used because oligonucleotides are too large for standard small molecule techniques, and they do not contain enough material to use classic methods to determine mass, such as using a balance.