Measuring nucleic acids by UV absorbance
DNA and RNA absorb ultraviolet light strongly at 260 nm, because of the ring structure of their bases. A spectrophotometer, such as a cuvette instrument or a microvolume device, reads this absorbance (A260) and turns it into a concentration. Readings at 280 nm and 230 nm then show whether proteins, salts or solvents are contaminating the sample.
The formulas
- Concentration (ng/µL, the same as µg/mL) = A260 × conversion factor × dilution factor
- Conversion factors for a 1 cm path: 50 for double-stranded DNA, 40 for RNA, about 33 for single-stranded DNA and oligonucleotides
- Total amount (µg) = concentration × volume (µL) ÷ 1,000
- Copies per µL = concentration (ng/µL) × 6.022 × 10²³ ÷ (length × 650 g/mol per base pair × 10⁹) for double-stranded DNA
Worked example
A plasmid prep is diluted 1:10 and reads A260 = 0.75, A280 = 0.40 and A230 = 0.36. The total volume is 50 µL.
- Concentration: 0.75 × 50 × 10 = 375 ng/µL.
- Total yield: 375 × 50 ÷ 1,000 = 18.75 µg.
- A260/A280 = 0.75 ÷ 0.40 = 1.88, good purity for DNA.
- A260/A230 = 0.75 ÷ 0.36 = 2.08, no sign of salt carry-over.
If the plasmid is 5,000 bp, that is about 6.9 × 10¹⁰ copies per µL.
Reading the purity ratios
| Ratio | Expected | Low values suggest |
|---|---|---|
| A260/A280, DNA | about 1.8 | Protein or phenol |
| A260/A280, RNA | about 2.0 | Protein or phenol |
| A260/A230 | 2.0 to 2.2 | Guanidine salts, EDTA, phenol, carbohydrates |
A DNA prep with an A260/A280 well above 2.0 may contain RNA; treating with RNase and re-purifying fixes that.
Getting reliable readings
- Stay in the linear range: absorbances between about 0.1 and 1.0 are most reliable on a cuvette instrument. Dilute strong samples.
- Blank with the same buffer the sample is in, such as TE or elution buffer.
- Mix well: genomic DNA is viscous and can give uneven readings.
- pH affects the 260/280 ratio: acidic water lowers it. Measuring in a slightly basic buffer gives more consistent values.
- Absorbance cannot tell DNA from RNA or free nucleotides. For low concentrations or mixed samples, fluorescent dye assays are more specific.