Oligo calculator
Converts a DNA oligo between nmol, µg and A260 units, and gives the resuspension volume.
Converts a DNA oligo between nmol, µg and A260 units, and gives the resuspension volume.
An oligo tube is labelled in nanomoles, a spectrophotometer reads absorbance, and a protocol asks for micrograms. Two numbers connect them: the molecular weight and the extinction coefficient at 260 nm, both of which follow from the sequence.
An A260 unit, also written OD unit, is the amount of material that gives an absorbance of 1.0 in 1 mL read through a 1 cm cell. It is an amount, not a concentration, which is why it converts straight to nanomoles.
Bases stacked on top of one another absorb less light than the same bases would apart, an effect called hypochromicity. Adding up single-base coefficients therefore overestimates ε by several percent. The nearest-neighbour sum used here walks along the sequence adding the coefficient of each adjacent pair, then subtracts the coefficient of every base that two pairs have in common. The base and pair values are the ones measured by Warshaw and Tinoco and refined since, the same set the oligo suppliers use.
| Base | ε₂₆₀ | Pair | ε₂₆₀ |
|---|---|---|---|
| dA | 15,400 | dAA | 27,400 |
| dC | 7,400 | dCG | 18,000 |
| dG | 11,500 | dGC | 17,600 |
| dT | 8,700 | dTT | 16,800 |
All sixteen pairs and all four bases are built in; the table shows four of each as a sample. Units are M⁻¹cm⁻¹.
Each figure is the mass of one nucleoside monophosphate residue in the chain, and the constant removes the phosphate that a 5 prime hydroxyl oligo does not have. Add 79.98 for a 5 prime phosphate. This is the anhydrous, free-acid weight; the sodium salt a supplier ships weighs about 22 g/mol more per phosphate, which is why a tube weighed on a balance does not match.
The M13 forward primer GTAAAACGACGGCCAGT, 17 nt and 52.9 % GC, has ε₂₆₀ = 172,500 M⁻¹cm⁻¹ and MW = 5,228.5 g/mol. A 25 nmol tube of it is 25 × 5228.5 ÷ 1000 = 130.7 µg and 25 × 172500 ÷ 10⁶ = 4.31 A260 units. One A260 unit is 10⁶ ÷ 172500 = 5.80 nmol, or 30.3 µg. To make a 100 µM stock, add 1000 × 25 ÷ 100 = 250 µL of TE.
| Use | Concentration |
|---|---|
| Stock after resuspension | 100 µM |
| PCR primer working stock | 10 µM |
| PCR primer in the reaction | 0.2–0.5 µM |
| qPCR primer in the reaction | 0.1–0.3 µM |
| Sequencing primer | 3.2–5 µM |
A 10 µM working stock is a 1 in 10 dilution of the 100 µM stock, which keeps the frozen stock untouched and the freeze-thaw cycles off it. Thedilution calculator handles anything less tidy, and theTm calculator gives the melting temperature of the same oligo. For the mass and moles of a long DNA or RNA rather than an oligo, usenucleic acid mass to moles.
Add ten microlitres of buffer for every nanomole on the tube. A 25 nmol synthesis needs 250 µL, a 50 nmol synthesis 500 µL. The rule works because 1 nmol in 10 µL is 100 µmol per litre, and it does not depend on the sequence at all. Spin the tube down before opening it, add TE or nuclease-free water, vortex and leave it for a few minutes to dissolve.
From the nearest-neighbour method: add up the coefficient of every adjacent pair of bases and subtract the coefficient of every base that is counted twice. It is more accurate than adding single-base values because stacked neighbours absorb less light than free bases do, and it is the method the oligo suppliers use.
Usually because the tube figure is the amount the supplier measured at synthesis and some of it stays on the walls, or because the oligo was not fully dissolved and the sample was read from the top of the tube. Vortex, spin and read again. A reading taken above A260 of about 1.0 is also outside the linear range of most spectrophotometers, so dilute and multiply back.
No. A standard synthetic oligo has a free hydroxyl at the 5 prime end, which is what the molecular weight here assumes. If the oligo was ordered with a 5 prime phosphate, for ligation, add 79.98 g/mol. Dyes, biotin and other modifications add their own mass and absorbance and are not covered.
No. The nearest-neighbour coefficients here are the DNA set, and the RNA values differ, so a sequence containing U is refused rather than converted with the wrong numbers. For the molar amount of a long RNA or DNA from its mass, use the nucleic acid mass to moles calculator instead.