How to measure the concentration, purity and yield of DNA and RNA — the spectrophotometric and fluorometric methods, what the ratios mean, and how to convert an absorbance reading into ng/µL. Calculators are built into the page.
Spectrophotometry (A260)
Nucleic acids absorb UV at 260 nm, and absorbance is proportional to concentration. Multiply A260 by the dilution and a type-specific factor (50 for dsDNA, 40 for RNA, 33 for ssDNA) to get ng/µL:
Concentration = A260 × dilution × factor (50 dsDNA, 40 RNA, 33 ssDNA), in ng/µL. Purity A260/A280 ≈ 1.8 is typical for pure DNA and ≈ 2.0 for RNA. Read absorbance in the linear range (A260 ~0.1–1.0).
It’s quick and needs no standards, but it can’t distinguish DNA from RNA or free nucleotides, and it’s unreliable below ~10 ng/µL.
What the purity ratios mean
- A260/A280: ~1.8 for pure DNA, ~2.0 for pure RNA. Lower values indicate protein or phenol carryover.
- A260/A230: 2.0–2.2 expected; low values point to guanidinium salts, phenol or carbohydrate contamination.
Fluorometry (Qubit and similar)
Dye-based fluorometric assays bind specifically to dsDNA, ssDNA or RNA, so they are far more specific and sensitive than A260 — the method of choice for low-concentration or precious samples (e.g. NGS library prep). They need a standard curve but ignore contaminating nucleotides and the wrong nucleic-acid type.
Cell density (OD600)
For bacterial cultures, optical density at 600 nm estimates cell number. Convert OD600 to cells/mL (adjust the factor for your strain and instrument):
Edit OD600 or cells/mL. Default factor 8×108 cells/mL per OD 1.0 is a common value for E. coli; it varies by strain, growth phase and spectrophotometer — calibrate for accurate work. Reliable only in the linear range (roughly OD < 0.8; dilute and re-read above that).
Which method when?
- Routine, sufficient material: A260 spectrophotometry is fast and adequate.
- Low concentration, NGS, or accuracy critical: use fluorometry.
- Always check purity ratios if downstream steps are sensitive (sequencing, transfection).
Frequently asked questions
How do I convert A260 to concentration?
concentration (ng/µL) = A260 × dilution × factor (50 dsDNA, 40 RNA, 33 ssDNA). Use the calculator above.
Spectrophotometer or Qubit?
Use a spectrophotometer for quick checks with ample material; use fluorometry for low concentrations or when specificity and accuracy matter.
What does a low A260/A230 mean?
Contamination by guanidinium salts, phenol or carbohydrates carried over from extraction; re-purify if it affects downstream steps.
