What does the A260/A280 ratio tell you?

It's a purity check for protein contamination. Nucleic acids absorb strongly at 260 nm and proteins absorb at 280 nm (from aromatic amino acids like tryptophan and tyrosine), so the ratio of the two reflects how clean the prep is. Pure DNA reads about 1.8 and pure RNA about 2.0. A ratio noticeably below those values (say under 1.7 for DNA) usually means residual protein or phenol from the extraction.

What does the A260/A230 ratio tell you?

It's a second purity check, sensitive to a different set of contaminants: chaotropic salts (like guanidine), phenol, carbohydrates, and EDTA, which all absorb around 230 nm. A clean sample reads about 2.0 to 2.2. A low A260/A230 (below about 1.8) flags leftover extraction reagents even when the 260/280 looks fine, which is why both ratios are reported together.

Why is an A260 of 1.0 equal to 50 ug/mL for DNA but 40 for RNA?

Because different nucleic acids absorb 260 nm light with slightly different efficiency per unit mass, based on their base composition and structure. Double-stranded DNA's bases are stacked and partially hidden (hypochromicity), so it takes more DNA to reach a given absorbance — about 50 ug/mL per A260 unit. Single-stranded DNA (33) and RNA (40) have more exposed bases and absorb more per microgram, so a lower concentration reaches the same absorbance.

Do I need to account for the dilution I made?

Yes. The A260 you read reflects the diluted sample, so the calculated concentration is the concentration of what's in the cuvette. To get back to your original undiluted stock, multiply by the dilution factor. For example, a 1:100 dilution reading A260 = 0.2 gives 0.2 x 50 x 100 = 1000 ug/mL for dsDNA. Forgetting the dilution factor is one of the most common quantification mistakes.

Is ng/uL the same as ug/mL?

Yes, they're numerically identical: 1 ug/mL equals 1 ng/uL. Instruments like the NanoDrop report ng/uL and older bench spectrophotometer protocols often use ug/mL, but the number is the same, so no conversion is needed between them.

DNA & RNA Concentration Calculator (A260/A280), Free

Sample type

A₂₆₀ (absorbance at 260 nm)

Dilution factor (1 if undiluted)

A₂₈₀ (for the 260/280 ratio)

A₂₃₀ (optional, for 260/230)

Concentration

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A260 / A280

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A260 / A230

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How the numbers work

Nucleic acids absorb ultraviolet light most strongly at 260 nm, and the amount of absorbance is proportional to concentration (Beer-Lambert law). Empirically, at a 1 cm path length an A₂₆₀ of 1.0 corresponds to about 50 µg/mL of double-stranded DNA, 33 µg/mL of single-stranded DNA, or 40 µg/mL of RNA — the differences come from how exposed the bases are (double-stranded DNA hides its stacked bases, so it takes more of it to reach the same absorbance). Multiply A₂₆₀ by the right factor and by any dilution you made, and you have the concentration. The two ratios are quick purity checks against different contaminants.

The two purity ratios, at a glance

Ratio	Clean value	If it's low, suspect…
A260 / A280	~1.8 (DNA), ~2.0 (RNA)	Residual protein or phenol (they absorb at 280 nm)
A260 / A230	~2.0 – 2.2	Chaotropic salts (guanidine), phenol, carbohydrates, EDTA (absorb at 230 nm)

Both ratios are reported together because they catch different problems — a prep can have a perfect 260/280 but a poor 260/230 if extraction salts weren't fully washed out.

Worked example (the default)

Double-stranded DNA, read at A260 = 0.400 after a 1:50 dilution A280 = 0.222, A230 = 0.182 Concentration: = A260 × factor × dilution = 0.400 × 50 µg/mL × 50 = 1000 µg/mL = 1000 ng/µL (1 µg/mL = 1 ng/µL) Purity: A260/A280 = 0.400 / 0.222 = 1.80 → ideal for DNA (clean) A260/A230 = 0.400 / 0.182 = 2.20 → in the clean 2.0–2.2 range

A textbook-clean genomic DNA prep. Drop the A280 to, say, 0.27 and the 260/280 falls to ~1.48, flagging protein or phenol carryover.

FAQ

What does A260/A280 tell you?
Protein contamination. Nucleic acids absorb at 260, proteins at 280. Pure DNA ≈ 1.8, pure RNA ≈ 2.0; noticeably lower means residual protein or phenol.

What does A260/A230 tell you?
A second check for salts (guanidine), phenol, carbohydrates, and EDTA, which absorb near 230. Clean is ~2.0–2.2; low flags leftover extraction reagents.

Why 50 for DNA but 40 for RNA?
They absorb 260 nm light differently per microgram. Double-stranded DNA's stacked bases are partly hidden (hypochromicity), so it takes ~50 µg/mL to reach A260 = 1; ssDNA (33) and RNA (40) have more exposed bases.

Do I include the dilution factor?
Yes — the A260 reflects the diluted sample, so multiply by the dilution factor to recover the original stock concentration. Forgetting it is a very common mistake.

Is ng/µL the same as µg/mL?
Yes, numerically identical (1 µg/mL = 1 ng/µL). NanoDrops report ng/µL; older protocols use µg/mL; same number.