Most manufacturers supply their oligonucleotides in a form that has been deprotected, desalted, to remove organic contaminants, and lyophilized. Further purification (i.e., HPLC or acrylamide gel purification) is usually NOT required. It is recommended that oligos be redissolved in a small volume (e.g., 100 microliters) of a sterile, buffered aqueous solution such TE buffer [10 mM Tris-HCl, 1 mM EDTA (pH 7.0)], as they may not readily dissolve in sterile distilled water. For long term storage, samples should be stored dry (lyophilized) at -20 C.
Quantitation of Oligonucleotides
Oligonucleotides, like double-stranded DNA, are most commonly quantified by measuring their absorbance of ultraviolet light at 260 nm. Single-stranded oligonucleotides (ssDNA) dissolved in neutral aqueous solution at a concentration of 33 mg/ml will have an absorbance at 260 nm (in a 1 cm cuvette) of approximately 1.0 unit, or optical density unit (OD unit). For comparison, an OD value of 1.0 corresponds to approximately 50 mg/ml for double-stranded DNA (dsDNA). To determine the concentration of an oligonucleotide (e.g., primer) stock solution, make one to a few dilutions of the stock solution (e.g., 100 - 500 fold) in distilled water or dilute TE buffer, sometimes referred to as "Te" [10 mM Tris-HCl, 0.1 mM EDTA (pH 7.0)], measure its (their) absorbance at 260 nm and calculate the concentration using the formula below:
where "A" is the measured absorbance, "33,000" is a conversion factor from mass to concentration, and "MW" is the molecular weight of the oligonucleotide, which can be calculated using the formula MW = (A x 312.2) + (C x 288.2) + (G x 328.2) + (T x 303.2) - 61. If you do not know the actual molecular weight of the particular oligonucleotide, substitute the term "N x 330" for MW, where "N" is the number of bases in the oligo, times the average molecular weight of a nucleotide. Other useful conversion information is given below:
pmoles/ mg = 106 / MW
pmoles/ OD = 109/ E260
mg/ OD = MW x 103 / E260
GC Content = (#G + #C / Total # bases) x 100
Numerous formulas* exist to determine the theoretical "melting temperature" or "Tm" of nucleic acids (as well as oligonucleotides) and these may serve as a starting point for determining annealing conditions for PCR applications. The Tm is the temperature at which half of the potential binding sites in a DNA are thought to have primer molecules bound to them. Longer primers, or ones with higher G+C content (number of guanosine and cytosine residues), will have higher Tm values - this is due to a greater number of hydrogen bonds per molecule. However, because of these and other considerations, it is best to determine and optimize (empirically) the annealing conditions by performing the reaction at several temperatures, starting approximately 5 C below any calculated Tm. The formula below can be used to estimate the melting temperature for oligonucleotides:
where [Na+] is the molar salt concentration; [K+] = [Na+] and n = number of bases in the oligonucleotide
Example: To calculate the melting temperature of a 22mer oligonucleotide with 60% G+C in 50mM KCl . . .
*An alternative formula, used by Operon Technologies (see Operon's Oligo Toolkit), is as follows: