Molecular Tools

Oligo Concentration Calculator from A₂₆₀ or Mass

Convert a NanoDrop reading or a weighed mass into molar concentration for any DNA oligonucleotide. The calculator derives sequence-specific extinction coefficient ε₂₆₀ and molecular weight, then applies the Beer–Lambert law.

Calculate oligo concentration

Paste your sequence and enter either an A₂₆₀ absorbance or a mass plus volume. The calculator reports µM, ng/µL, and conversion factors specific to the sequence.

Quick presets

Oligonucleotide sequence

DNA sequence (5′ → 3′)

Length: 20 ntMW: 5.99 kDaε₂₆₀: 183.5 × 10³ M⁻¹cm⁻¹

A₂₆₀ reading

Absorbance at 260 nm

NanoDrop reading at 1 cm equivalent path length. Typical primer stocks: 0.4–3.0.

Path length

Cuvette path (cm)

Standard cuvette: 1 cm. NanoDrop reports normalised to 1 cm by default.

Concentration

6.54 µM

Mass concentration

39.18 ng/µL

Molar (M)

6.54e-6 M

Picomolar

6.54e+6 pM

Length

GC content

55.0

Mol. weight

5.99

kDa

ε₂₆₀

183.5

× 10³ M⁻¹cm⁻¹

Calculation

Beer–Lambert law

A = ε · c · l → c = A / (ε · l)

c = 1.2 / (183,483 × 1)

c = 6.540e-6 M = 6.54 µM

Useful conversions for this oligo

1 OD₂₆₀ =5.5 nmol

1 OD₂₆₀ =32.65 µg

1 µg =166.92 pmol

1 nmol =5.99 µg

Figure 1. Spectrophotometric quantification of a DNA oligonucleotide through the Beer–Lambert law. Ultraviolet light at 260 nm passes through a cuvette of path length l containing oligonucleotide at concentration c. Absorbance A equals the logarithm of the ratio of incident to transmitted intensity, and scales linearly with concentration through the molar extinction coefficient ε₂₆₀. The coefficient is sequence-specific: nearest-neighbor calculations weighted by dinucleotide stacking yield values accurate within ±3% for unmodified DNA between 8 and 100 nucleotides.

Beer–Lambert law for oligonucleotides

August Beer and Johann Heinrich Lambert worked out the linear absorbance relationship in the 19th century. Their law states that absorbance equals the molar extinction coefficient ε multiplied by concentration c and path length l: A = ε · c · l. Rearranging gives c = A / (ε · l), the working equation for concentration from any absorbance reading.

Nucleic acids absorb strongly at 260 nm because of π-π* electronic transitions in the aromatic purine and pyrimidine bases. Each base contributes a characteristic ε: adenine 15,200, thymine 8,400, guanine 12,010, cytosine 7,050 M⁻¹cm⁻¹. A naive sum overestimates the actual oligonucleotide ε because adjacent bases stack and shield each other from incident light, an effect called hypochromicity. The nearest-neighbor correction subtracts this stacking contribution by averaging dinucleotide-step extinctions.

A practical consequence: two oligos of the same length but different sequence can differ in ε by 20% or more. An AT-heavy 20-mer might have ε near 220,000, while a GC-heavy 20-mer of the same length stays closer to 170,000. The generic "1 OD = 33 µg/mL" rule, which assumes dsDNA, fails badly for short ssDNA. Sequence-specific ε is necessary for any quantitative work.

How NanoDrop and spectrophotometers measure A₂₆₀

Conventional UV-Vis spectrophotometers use 1 cm path-length quartz cuvettes with 50–200 µL sample volumes. The instrument compares transmitted light through the sample against a buffer blank and reports absorbance directly. NanoDrop instruments shrink the sample to 1–2 µL by exploiting surface tension between two optical pedestals, with a 0.05–1 mm variable path length. The displayed A₂₆₀ is normalised to 1 cm equivalent, so the same Beer–Lambert arithmetic applies regardless of which platform produced the reading.

Working concentrations sit between A₂₆₀ = 0.1 and 2.0 for accurate quantitation. Below 0.1, instrument noise dominates and readings carry 10–20% relative error. Above 2.0, the linear dynamic range breaks down because too little light reaches the detector. Dilute concentrated stocks 1:10 before reading if A₂₆₀ exceeds 2.0; the working dilution returns to the linear range and the result scales back by the dilution factor.

Worked examples

Example 1: NanoDrop reading of a primer stock

Sequence: 5′-ACCACAGTCCATGCCATCAC-3′ (20 nt). NanoDrop reads A₂₆₀ = 1.2 at 1 cm equivalent path.

Calculated ε₂₆₀ for this sequence ≈ 196,800 M⁻¹cm⁻¹ (from nearest-neighbor sum).

c = 1.2 / (196,800 × 1) = 6.1 × 10⁻⁶ M = 6.1 µM

Mass concentration: 6.1 µM × 6,135 g/mol ÷ 1000 = 37 ng/µL. This stock matches a typical resuspended PCR primer; dilute 1:60 in TE to reach a 100 nM working stock.

Example 2: Mass-based quantification

A 30-mer ssDNA antisense oligo. QC sheet reports 50 µg mass in 100 µL of TE buffer. Sequence-derived MW = 9,250 g/mol.

c = mass / (MW × V) = 50 × 10⁻⁶ g / (9,250 g/mol × 100 × 10⁻⁶ L) = 54 × 10⁻⁶ M = 54 µM

Total moles = 50 µg / 9,250 g/mol = 5.4 nmol. Verify by A₂₆₀ measurement and compare against this calculation. A discrepancy above 10% indicates incomplete resuspension or a yield reporting error.

When to use A₂₆₀ versus mass calculation

A₂₆₀ wins for routine bench quantification. The measurement takes 30 seconds, requires 1–2 µL of sample, and integrates over the full ε of the oligonucleotide. Resuspended stocks and working dilutions are quickest to verify this way. The downside: any UV-absorbing contaminant (modified bases, fluorophores, residual ammonia from synthesis cleavage) inflates the reading.

Mass-based calculation matters when you start from a known weighed amount, typically the vendor-reported yield on the QC sheet. This approach is independent of any spectroscopic interference but assumes the oligo is fully soluble and that the reported mass is accurate to within 5%. For modified oligos with fluorophores or other chromophores at 260 nm, mass-based is often more reliable than A₂₆₀.

For absolute accuracy, run both. A 10% agreement between the two methods confirms that the stock matches expectations. Larger discrepancies point to insoluble pellet residue, vendor yield miscalculation, or chromophore contamination.

Quality indicators in absorbance ratios

Modern spectrophotometers report A₂₆₀, A₂₈₀, and A₂₃₀ simultaneously. The ratios flag contamination types. A₂₆₀/A₂₈₀ near 1.8–2.0 indicates clean nucleic acid; below 1.7 suggests protein carryover (proteins absorb at 280 nm via tryptophan and tyrosine). A₂₆₀/A₂₃₀ above 2.0 confirms absence of guanidinium, phenol, or carbohydrate contamination from silica-column extraction.

Synthetic oligonucleotides shipped lyophilised typically score A₂₆₀/A₂₈₀ around 1.95–2.05 and A₂₆₀/A₂₃₀ above 2.2. Values outside these ranges point to a problem with the synthesis or with how you resuspended the pellet. For genomic DNA preparations the ratios diagnose nuclease purification quality; for oligonucleotides they diagnose synthesis cleavage and deprotection completeness.

Limitations and caveats

Sequence-derived ε₂₆₀ values assume only Watson–Crick A, T, G, and C residues at neutral pH. Modified bases such as inosine, 5-methylcytosine, locked nucleic acid, or 2′-O-methyl RNA shift the extinction coefficient by 2–10%. Fluorophore labels add discrete chromophore contributions that must be subtracted from the total absorbance before applying Beer–Lambert.

At low concentrations (below 10 nM), polypropylene tube adsorption removes a measurable fraction of oligo from solution. The A₂₆₀ reading reflects what stays in solution, not what was originally pipetted. Carrier nucleic acid (10 ng/µL tRNA) or low-binding tubes prevent this loss for sub-nanomolar work.

This calculator is intended for teaching and routine lab use. Pharmaceutical and clinical quantitation should use validated HPLC or LC-MS methods alongside spectroscopy. The IDT OligoAnalyzer and similar commercial tools cross-check ε values against independently calibrated reference standards.

Frequently asked questions

How do I convert A260 to oligo concentration?

Apply Beer–Lambert: concentration (M) equals absorbance divided by the product of extinction coefficient and path length. For DNA oligonucleotides, the extinction coefficient at 260 nm depends on the sequence because adjacent bases stack and reduce light absorption (hypochromicity). A typical 20-mer has ε around 200,000 M⁻¹cm⁻¹, so an A₂₆₀ reading of 1.0 in a 1 cm cuvette gives 5 µM. NanoDrop instruments normalise to a 1 cm equivalent path length automatically, so the same arithmetic applies regardless of the actual 1 mm pedestal gap.

Why is the 1 OD = 33 µg/mL rule inaccurate for short oligos?

The 33 µg/mL approximation comes from double-stranded DNA at neutral pH and assumes 50 µg/mL would give A₂₆₀ = 1.5. Single-stranded oligonucleotides absorb more strongly per base than dsDNA because the hypochromic effect of base pairing is absent. The actual conversion runs around 25–35 µg/mL per OD for ssDNA, and varies by 30% across sequences because A, T, G, and C have different molar extinctions (A = 15,200; T = 8,400; G = 12,010; C = 7,050 M⁻¹cm⁻¹). For accurate quantitation, use the sequence-specific ε rather than a generic factor.

What is the extinction coefficient and how is it calculated?

The molar extinction coefficient ε₂₆₀ is the proportionality constant between absorbance and concentration in the Beer–Lambert law. For oligonucleotides, the nearest-neighbor method sums the extinction of each dinucleotide step along the sequence and subtracts the contribution of internal monomers to correct for double-counting. This method, refined by Cantor, Warshaw, and Shapiro in 1970, accounts for base stacking and produces values accurate to ±3% for unmodified DNA between 8 and 100 nt. The IDT online calculator and most commercial tools implement the same algorithm.

Why does my A260 reading differ from the QC sheet value?

Three common causes: incomplete resuspension, salt contamination, or instrument calibration drift. Re-vortex and spin the stock, then re-measure. High salt from incomplete desalting raises baseline absorbance non-specifically; a clean blank with the same buffer resolves this. Verify the NanoDrop by measuring a fresh A₂₆₀ = 1.0 reference standard. If the discrepancy persists beyond 10%, the supplier may have over- or underestimated yield during QC. Most vendors will recalculate or replace orders on request.

How do I quantify a modified oligo (FAM, biotin, LNA)?

Fluorophores and quenchers absorb at 260 nm and inflate the apparent oligo concentration. FAM contributes about 20,000 M⁻¹cm⁻¹ at 260 nm; Cy5 adds 10,000. Subtract the modifier ε from the total A₂₆₀ reading before applying the standard calculation, or measure the modifier separately at its own λmax (FAM at 495 nm) and back-calculate. For LNA and 2′-O-methyl modifications, the ε is similar to the corresponding unmodified base, so the standard calculation works within 5%. The vendor QC sheet usually lists the corrected ε.

What buffer is best for A260 measurement?

Low-salt TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) or nuclease-free water give the cleanest A₂₆₀ readings. EDTA does not absorb at 260 nm at this concentration; Tris contributes negligibly. Avoid phosphate buffers above 50 mM because they shift the baseline. For NanoDrop measurements, use the same buffer for the blank as for the sample to subtract any minor background. Air-bubble-free pipetting matters because bubbles inflate apparent absorbance by 10–30%.

Why is A260/A280 ratio reported alongside concentration?

The A₂₆₀/A₂₈₀ ratio flags protein or phenol contamination. Pure DNA gives 1.8–2.0; pure RNA gives 2.0–2.2. Values below 1.7 suggest residual protein from organic extraction. Values below 1.5 indicate substantial contamination that will inhibit PCR or sequencing. The A₂₆₀/A₂₃₀ ratio (above 2.0 for clean samples) flags carbohydrate or guanidinium contamination from silica column extractions. Pure synthetic oligonucleotides typically score 1.95–2.05 at A₂₆₀/A₂₈₀ because they contain no protein.

References. Nearest-neighbor extinction coefficients follow Cantor, Warshaw, and Shapiro 1970. Spectrophotometric method overview available from NCBI.