Oligonucleotide Resuspension Calculator
Oligonucleotide Resuspension Calculator
How much buffer a lyophilised oligo needs to reach a target stock concentration — and why the OD₂₆₀ figure on the tube converts to micrograms and nanomoles differently for every oligo you order.
Oligonucleotide Resuspension
nmol, target µM → µL45.2 nmol delivered on the tube, to be resuspended as a 100 µM stock
Formula
and in reverse: concentration (µM) = nmol × 1000 ÷ volume (µL)
an oligo at C µM contains C pmol per µL — the two are the same number
- the ×1000
- the unit conversion, and worth deriving once. A target of C µM is C µmol/L. The n nanomoles you have is n × 10⁻⁹ mol, so the volume is n × 10⁻⁹ ÷ (C × 10⁻⁶) litres = n/C millilitres = 1000 × n/C microlitres. Nothing about the oligo enters it
- why nmol is the useful unit
- because it is an amount of substance, so it converts into a molar concentration without knowing anything else. A figure in micrograms or OD units does not: to get from either of those to nanomoles you need the oligo's molar mass or its extinction coefficient, both of which depend on its sequence
- µM and pmol/µL
- identical numbers. A 10 µM working solution is 10 pmol/µL, so 0.5 µL of it delivers 5 pmol — which in a 25 µL reaction is 0.2 µM. The final concentration that belongs in a given assay is whatever that assay was validated at
- an A₂₆₀ unit
- the amount of material that gives an absorbance of 1.0 at 260 nm in 1 mL with a 1 cm path. It converts to nanomoles as 10⁶ ÷ ε₂₆₀, where ε is the oligo's molar extinction coefficient in L mol⁻¹ cm⁻¹, and to micrograms by multiplying that by the molar mass and dividing by 1,000
- the 33 µg per A₂₆₀ unit rule
- THE POINT OF THE SECOND HALF OF THE PAGE: a convention, not a measurement of your oligo. It is the conventional single-stranded DNA factor, alongside 50 for double-stranded DNA and 40 for RNA. The direct measurement of the same quantity recommends 38 µg per A₂₆₀ unit for non-repetitive single-stranded DNA at 40–80% GC — and for a short oligo the right figure depends on base composition and spans roughly twofold
Worked example
45.2 nmol delivered on the tube, to be resuspended as a 100 µM stock
Volume = 45.2 × 1000 ÷ 100 = 452.0 µL of TE or nuclease-free water
Check it: 45.2 nmol in 452 µL is 0.1 nmol/µL, which is 100 pmol/µL, which is 100 µM
The same 45.2 nmol taken straight to a 10 µM working stock would need 4,520 µL — which is why nobody does that. Make the concentrated stock, then dilute 10 µL into 90 µL for 10 µM
At 10 µM, 0.5 µL per reaction delivers 5 pmol, or 0.2 µM in a 25 µL reaction. What that final figure should be is set by the assay's validation, not by a rule
Now the part the tube does not make obvious. If the certificate had quoted only OD₂₆₀ units, converting to nanomoles needs the oligo's extinction coefficient — and for a 20-mer that ranges from about 142,000 to 301,000 L mol⁻¹ cm⁻¹ depending on base composition alone
In micrograms per A₂₆₀ unit, that same 20-mer spans roughly 20.6 to 40.3 µg between composition extremes. The 33 µg rule of thumb sits inside that spread and is not a substitute for the figure on the certificate of analysis
Buffer volume for a 100 µM stock
| Delivered (nmol) | Volume for 100 µM (µL) | Volume for 10 µM (µL) |
|---|---|---|
| 5 | 50.0 | 500.0 |
| 10 | 100.0 | 1,000.0 |
| 25 | 250.0 | 2,500.0 |
| 45.2 | 452.0 | 4,520.0 |
| 100 | 1,000.0 | 10,000.0 |
Why micrograms per A₂₆₀ unit is not one number for oligos
| 20-mer | Molar mass (g/mol) | ε₂₆₀ (L mol⁻¹ cm⁻¹) | nmol per A₂₆₀ unit | µg per A₂₆₀ unit |
|---|---|---|---|---|
| All dA | 6,202 | 301,200 | 3.32 | 20.6 |
| All dG | 6,522 | 243,600 | 4.11 | 26.8 |
| Five of each base | 6,117 | 214,500 | 4.66 | 28.5 |
| All dT | 6,022 | 171,200 | 5.84 | 35.2 |
| All dC | 5,722 | 142,000 | 7.04 | 40.3 |
The conventional absorbance factors, and what they are
| Species | µg/mL per A₂₆₀ of 1.0 | Status |
|---|---|---|
| Double-stranded DNA | 50 | The long-standing laboratory convention |
| Single-stranded DNA | 33 | The long-standing laboratory convention |
| RNA | 40 | The long-standing laboratory convention |
| Single-stranded DNA, measured | 38 | Recommended from direct measurement, for non-repetitive sequence at 40–80% GC |
| Single-stranded RNA, measured | 37 | Recommended from the same direct measurement |
| A specific oligonucleotide | Its own figure | From the certificate of analysis, or computed from its base composition — the only one of these that is about your tube |
The easy half and the half that catches people
The resuspension itself is the easiest calculation in molecular biology, and it is easy for a specific reason: the tube is labelled in nanomoles. Nanomoles are an amount of substance, and a target concentration in micromolar is an amount of substance per volume, so the volume follows without knowing anything at all about the oligo. Multiply the nanomoles by a thousand, divide by the target in micromolar, and the answer is microlitres. Forty-five point two nanomoles to a hundred micromolar is 452 microlitres, and the check is immediate: 45.2 nmol in 452 µL is 0.1 nmol per microlitre, which is 100 pmol per microlitre, which is 100 µM. The convention of making stocks at 100 µM exists because it dilutes tenfold to a 10 µM working solution, and because a concentrated stock survives storage better than a dilute one — adsorption to plastic takes roughly a fixed amount rather than a fixed fraction, so it hurts a dilute solution more.
Two practical points sit around the arithmetic rather than in it. Spin the tube before opening it: lyophilised oligonucleotide is a film, not a pellet, and it redistributes in transit so that a noticeable share ends up under the cap. And give it time to dissolve, with a vortex and a few minutes standing, because an oligo that has only partly redissolved yields a stock that is weaker than calculated at first and slowly strengthens as the rest goes into solution — which looks exactly like a pipetting problem and is not one.
The half that catches people is the other conversion: from A₂₆₀ units, or micrograms, to nanomoles. Three factors are in general use — 50 µg/mL per absorbance unit for double-stranded DNA, 33 for single-stranded DNA and 40 for RNA — and they are conventions rather than constants. They are perfectly serviceable for long, mixed-sequence nucleic acid, which is the job they were meant for. They are not serviceable for a 20-mer. Absorbance at 260 nm comes from the bases, and the bases differ: the revised molar extinction coefficients are 15.06 for dAMP, 12.18 for dGMP, 8.56 for dTMP and 7.10 for dCMP in litres per millimole per centimetre, a spread of more than two to one. Summing those across a 20-mer and dividing gives, for the composition extremes, anything from about 20.6 to 40.3 micrograms per absorbance unit. The same absorbance reading therefore corresponds to a twofold range of mass depending only on what the oligo is made of.
It is worth being precise about how good the 33 is, because it is better than that arithmetic makes it look and worse than it is usually treated. Summing monomer coefficients overstates a real single strand's absorbance, because stacked bases absorb less than free ones, so the computed micrograms-per-unit figures above are all a few per cent low — which is why an equal-composition 20-mer computes to 28.5 rather than sitting on 33. Meanwhile the most careful direct measurement of the quantity concluded that nearly all previously published nucleotide extinction coefficients were too large, by as much as 7%, and recommended 38 micrograms per absorbance unit for non-repetitive single-stranded DNA at 40 to 80% GC — not 33. So the conventional figure is bracketed rather than confirmed, and for a specific short oligo it was never the right tool anyway. The manufacturer computes a weight-per-OD and an nmol-per-OD for the sequence you ordered and prints both on the certificate of analysis; the standard protocol is to use them. When no certificate is to hand, compute the extinction coefficient from the base composition rather than reaching for a generic factor, and treat the result as an estimate with a few per cent of slack in it.
Frequently asked questions
How much buffer do I add to resuspend an oligo?
Volume in microlitres = nanomoles × 1000 ÷ the target concentration in micromolar. For 45.2 nmol at 100 µM that is 452 µL. The calculation needs nothing about the oligo itself, because nanomoles are an amount of substance and micromolar is an amount per volume.
Why is 100 µM the usual stock concentration?
Because it dilutes one part in ten to a 10 µM working solution, and because concentrated stocks store better. Adsorption to plastic removes roughly a fixed amount rather than a fixed fraction, so it costs a dilute solution proportionally more, and repeated freezing and thawing does relatively more damage to a dilute stock.
How do OD₂₆₀ units, micrograms and nanomoles relate for an oligo?
One A₂₆₀ unit is the material giving an absorbance of 1.0 in 1 mL at a 1 cm path. It equals 10⁶ ÷ ε₂₆₀ nanomoles, where ε is that oligo's molar extinction coefficient, and multiplying by the molar mass converts to micrograms. Both ε and the molar mass depend on the sequence, which is why the certificate of analysis quotes them per oligo.
Is 33 µg per A₂₆₀ unit accurate for a primer?
It is a convention for single-stranded DNA in general, not a figure for your oligo. For a 20-mer the value ranges from about 20.6 to 40.3 µg per absorbance unit on base composition alone. A direct measurement of the same quantity recommends 38 µg per unit for non-repetitive single-stranded DNA at 40 to 80% GC, so even the general figure is disputed.
How do I make a 10 µM working dilution from a 100 µM stock?
One part stock to nine parts buffer — 10 µL into 90 µL. At 10 µM, 0.5 µL delivers 5 pmol, which is 0.2 µM in a 25 µL reaction. The final concentration your assay needs is whatever it was validated at; keep the concentrated stock for storage and let the working dilution take the freeze–thaw cycles.
Why has my oligo stock come out weaker than calculated?
Usually because it has not fully dissolved, or because material was lost from the cap. Lyophilised oligo is a film that redistributes in transit, so spin the tube before opening it, and allow time with vortexing before use. A partly dissolved oligo gives a stock that starts weak and strengthens over days as the rest goes into solution.
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References
- Cavaluzzi MJ, Borer PN. Revised UV extinction coefficients for nucleoside-5′-monophosphates and unpaired DNA and RNA. Nucleic Acids Res. 2004;32(1):e13.
- Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press; 2001 — appendix on spectrophotometric quantification and the conventional absorbance factors.
- Thermo Fisher Scientific. Oligonucleotide protocols: resuspension, quantification and storage — on using the weight-per-OD and nmol-per-OD values from the certificate of analysis.
- Bustin SA, Benes V, Garson JA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611–622 — on reporting primer and probe concentrations.
Medical Disclaimer: The tools and content provided here are for educational and reference purposes only. They are not intended to substitute for professional medical advice, diagnosis, or treatment. Clinical decisions should always be based on the comprehensive assessment of a qualified healthcare professional.