How to calculate DNA concentration from A260 absorbance

How to · Updated 2026-11-10

Turn an A260 reading into ng/µL, check purity with 260/280 and 260/230, and get nM, copies per µL and a dilution, with a worked plasmid example.

DNA concentration in ng/µL is the background-corrected A260 reading times 50 for double-stranded DNA, times any dilution, divided by the path length in cm. The nucleic acid concentration calculator does that for one sample or a whole batch, and adds the purity ratios, molarity, copy number and the volumes for a dilution.

What you need

• Absorbance at 260 nm (A260) and 280 nm (A280). A230 and A320 make the result better if your instrument gives them.
• What you measured: double-stranded DNA, single-stranded DNA or RNA. Each has its own factor.
• Any dilution you made before reading, and the path length. Microvolume readers usually report values already scaled to 1 cm.
• For molarity or copies, the length of the DNA in base pairs and whether it is linear or a circular plasmid.

Step by step

1. Pick the sample type. On the tool, choose double-stranded DNA. The factor box fills in 50 (33 for single-stranded DNA, 40 for RNA). Change it only if you have a better factor, such as the one on an oligo’s data sheet.
2. Set the dilution and path. Type how many times you diluted the sample before reading, and the path length in cm.
3. Enter the readings. One row per sample: a name, A260, A280, then A230 and A320 if you have them. You can paste rows from a spreadsheet or open a CSV file.
4. Give the size. Type the length in bp and choose linear or circular. If you paste the exact sequence instead, its own mass is used.
5. Set a dilution target (optional). Choose a target in ng/µL, nM or copies/µL, and a final volume. The table then lists the stock and diluent volumes for each sample.

The maths the tool runs is the Beer–Lambert law with a fixed factor:

c (ng/µL) = (A260 − A320) × factor × dilution ÷ path length (cm)

Purity ratios use the same background correction: 260/280 = (A260 − A320) ÷ (A280 − A320), and likewise for 260/230. Then:

nM = c (ng/µL) ÷ molar mass (g/mol) × 10⁶ copies/µL = c × 10⁻⁹ × 6.022 × 10²³ ÷ molar mass

To make a working dilution by hand, the dilution calculator uses the same C₁V₁ = C₂V₂ rule. For any other molecule, the Beer–Lambert calculator handles absorbance with your own extinction coefficient.

A worked example

You diluted a plasmid miniprep 1 in 5 and read it in a 1 cm cuvette: A260 0.620, A280 0.335, A230 0.300 and A320 0.012. The plasmid is 5,400 bp and circular.

Background-corrected A260 = 0.620 − 0.012 = 0.608. Concentration = 0.608 × 50 × 5 ÷ 1 = 152 ng/µL.

Purity:

• 260/280 = 0.608 ÷ (0.335 − 0.012) = 0.608 ÷ 0.323 = 1.88
• 260/230 = 0.608 ÷ (0.300 − 0.012) = 0.608 ÷ 0.288 = 2.11

Both sit in the usual range for clean DNA, so the tool leaves the note as OK.

For a 5,400 bp circle, the tool’s molar mass from the average base pair mass is about 3,336,655 g/mol. So:

• Molarity = 152 ÷ 3,336,655 × 10⁶ ≈ 45.6 nM
• Copies = 152 × 10⁻⁹ × 6.022 × 10²³ ÷ 3,336,655 ≈ 2.74 × 10¹⁰ copies/µL

To make 50 µL at 20 ng/µL, you need 20 × 50 ÷ 152 = 6.58 µL of stock plus 43.4 µL of water or buffer.

Check your answer

• One A260 unit of double-stranded DNA is about 50 ng/µL, so an A260 of 0.6 in a 1 in 5 dilution should give a stock of roughly 0.6 × 50 × 5 = 150 ng/µL. If your number is ten times off, look at the dilution factor first.
• Readings below about 0.02 absorbance units are too faint to trust; readings far above the instrument’s linear range should be diluted and read again.
• A 260/280 well below 1.7 hints at protein or phenol; a 260/230 below 1.8 hints at salt or guanidine from the kit. A ratio above 2.0 for DNA can mean RNA is present.

Questions people ask

Why is the factor 50 for DNA but 40 for RNA? The strands absorb differently. Paired bases in a double helix absorb less than free single strands, so double-stranded DNA needs more mass to reach one absorbance unit.

Can I trust A260 for a PCR product? It counts all nucleic acid, including leftover primers and free nucleotides. Clean the product up first, or use a dye-based assay that only sees double-stranded DNA.

Do I need A320 at all? No, but it removes background from bubbles, dust or a cloudy sample. Without it, the tool assumes zero background.