DNA Concentration Calculator

Results

How to Use the DNA Concentration Calculator

1. 1. Choose the sample type. dsDNA (plasmids, PCR products, genomic DNA), ssDNA, RNA or a short oligonucleotide. This sets the conversion factor and, later, the molecular weight.
2. 2. Enter the readings. Type A₂₆₀. Add A₂₈₀ and A₂₃₀ to get the purity ratios, and a background reading (A₃₂₀) if your protocol subtracts one. Set the path length and the dilution factor if they are not 10 mm and 1.
3. 3. Optional: add a volume and a length. The volume gives total yield in µg; the length (bp for dsDNA, nt otherwise) adds molarity and pmol.
4. 4. Read the result. The concentration, the purity verdicts and the full working appear together. Switch to the ng ↔ pmol mode to convert an amount of DNA without any absorbance data.

How A260 Becomes a Concentration

Nucleic acids absorb ultraviolet light most strongly at 260 nm. By the Beer–Lambert law, the absorbance is proportional to concentration, so one conversion factor turns a reading into a mass concentration:

The factor ε is specific to the type of nucleic acid. Thermo Fisher's NanoDrop FAQ lists these values, in ng·cm/µL, and a New England Biolabs technical note gives the same three as µg/mL for a 10 mm path:

These are averages for a typical base composition. Short oligonucleotides vary with their sequence — an oligo's own extinction coefficient from the manufacturer is more accurate — and some references list a different value for ssDNA, such as 37 µg/mL. Use the override field if your protocol or supplier specifies another factor.

Reading the Purity Ratios

Proteins absorb near 280 nm and many common contaminants absorb near 230 nm, so two ratios give a quick check on what else is in the tube:

• A₂₆₀/A₂₈₀. About 1.8 for clean DNA (NEB reports 1.85–1.88 in buffer) and about 2.0–2.1 for RNA. A low value points to protein or phenol; a high value in a DNA prep can mean RNA carry-over. Acidic solutions read lower and basic ones higher, so check the pH of the buffer.
• A₂₆₀/A₂₃₀. A more sensitive indicator, usually 2.0–2.4 for clean samples. Guanidine salts, EDTA, detergents, phenol and carbohydrate lower it. NEB cautions that this ratio varies more than A₂₆₀/A₂₈₀ and should be read with care.
• The common threshold. In everyday practice both ratios above 1.8 count as clean enough for most downstream work.

Worked Example: A Plasmid Miniprep

A 5,000 bp plasmid is eluted in 50 µL, diluted 1 in 10 and read in a 10 mm cuvette: A₂₆₀ = 0.412, A₂₈₀ = 0.222, A₂₃₀ = 0.18. These are the calculator's default inputs.

1. Concentration: 0.412 × 50 × (10 ÷ 10) × 10 = 206 ng/µL.
2. Purity: 0.412 ÷ 0.222 = 1.86 and 0.412 ÷ 0.18 = 2.29. Both sit comfortably in the clean range.
3. Yield: 206 ng/µL × 50 µL = 10.3 µg of DNA in the tube.
4. Molarity: 206 × 1,000 ÷ (660 × 5,000) = 0.06242 pmol/µL = 62.42 nM, which is 3.121 pmol in the tube.

Converting Between ng and pmol

Cloning and ligation recipes are written in picomoles, but a spectrophotometer gives nanograms. The conversion needs the length and an average molecular weight per base pair or nucleotide:

The familiar check: 1 µg of a 1,000 bp fragment is 1.515 pmol (3.03 pmol of ends), the same as the 1.52 pmol per µg per kilobase figure that suppliers quote. The 660 g/mol per base pair is an average; some sources use 650, which changes pmol by about 1.5%. For an exact oligo molecular weight, use the manufacturer's value instead. To plan an insert-to-vector ratio from these amounts, use the Ligation Calculator.

Common Mistakes

• Forgetting the dilution factor. The cuvette value is the diluted sample; multiply back to get the stock.
• Using the dsDNA factor for an oligo or RNA. The difference is 33–50% in the answer.
• Reading outside the linear range. Keep A₂₆₀ between roughly 0.1 and 1.5; dilute concentrated samples and read again.
• Trusting absorbance for everything. Free nucleotides, RNA and single-stranded fragments all absorb at 260 nm, so absorbance overstates the amount of intact double-stranded DNA in a crude prep. Fluorescent dyes specific to dsDNA are more selective when that matters.

Frequently Asked Questions

How do you calculate DNA concentration from A260?

Multiply the A260 reading by the conversion factor — 50 ng/µL for double-stranded DNA, 33 for single-stranded DNA, 40 for RNA — and by any dilution factor. An A260 of 0.412 for dsDNA diluted 1 in 10 gives 0.412 × 50 × 10 = 206 ng/µL. For a path other than 10 mm, scale by 10 divided by the path length in millimetres.

What is a good A260/A280 ratio?

About 1.8 for pure DNA and about 2.0 for pure RNA. NEB reports 1.85–1.88 for clean dsDNA in buffer and about 2.1 for RNA. Ratios clearly below these suggest protein or phenol contamination; the ratio is also shifted by buffer pH, so read it as a guide and not a pass-or-fail test.

What does a low A260/A230 ratio mean?

Something other than nucleic acid is absorbing at 230 nm. The usual culprits are guanidine salts from column kits, EDTA, detergents, phenol and carbohydrate carry-over. Re-elute or re-precipitate the sample, and make sure the instrument was blanked with the same buffer as the sample.

How do you convert ng of DNA to pmol?

Divide the mass in nanograms by the molecular weight, which is about 660 g/mol per base pair for dsDNA, and multiply by 1,000. For 100 ng of a 3,000 bp plasmid: 100 × 1,000 ÷ (660 × 3,000) = 0.0505 pmol. The converter above does this in both directions.

Is ng/µL the same as µg/mL?

Yes. One nanogram per microlitre is 10⁻⁹ g per 10⁻⁶ L, which is 10⁻³ g/L or 1 µg/mL. That is why the A260 conversion factors are quoted both ways, as 50 ng/µL or 50 µg/mL per absorbance unit.

Why does my NanoDrop reading differ from a Qubit?

Absorbance counts anything that absorbs at 260 nm — RNA, free nucleotides and contaminants — while a fluorescent dye assay such as Qubit binds double-stranded DNA specifically. Absorbance is usually the higher number on crude preps. The two agree closely on clean, purified dsDNA.

My A260 is above 1.5. Can I still use it?

Dilute the sample and read again. Most spectrophotometers lose linearity at high absorbance, so a reading far above 1.5 underestimates the real concentration. Enter the dilution factor so the calculator scales the answer back to the stock.