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.
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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)
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.
Sources and how to cite this page
The conversion factors are the conventional ones used throughout molecular biology and tabulated in Molecular Cloning: A Laboratory Manual (Sambrook & Russell): at a 1 cm path length, one A260 unit corresponds to about 50 µg/mL of double-stranded DNA, 40 µg/mL of RNA, and 33 µg/mL of single-stranded DNA. The difference comes from the hyperchromic effect: unstacked single-stranded bases absorb more per unit mass than base-paired duplex.
Reading the purity ratios. An A260/A280 near 1.8 is the usual target for DNA and near 2.0 for RNA. A low ratio suggests protein or phenol carryover. The A260/A230 ratio is the more sensitive one for guanidine, phenol, and carbohydrate contamination, and should be roughly 2.0 to 2.2. These are conventions rather than hard thresholds, and they are pH and buffer sensitive, so read them as flags rather than verdicts.