UV-Vis spectrophotometry
Quantify DNA or RNA from the 260 nm base peak, using 50 ng/µL per absorbance unit for dsDNA, 40 for RNA and 33 for single-stranded DNA at a 1 cm path, and judge the prep from the A260/A280 and A260/A230 ratios.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
A260 nucleic acids
Quantify DNA or RNA from the 260 nm base peak, using 50 ng/µL per absorbance unit for dsDNA, 40 for RNA and 33 for single-stranded DNA at a 1 cm path, and judge the prep from the A260/A280 and A260/A230 ratios.
How it differs from the standard method
- Blank on the elution buffer, not water: Tris, EDTA and carried-over salts all absorb below 240 nm and shift A260/A230.
- A260 cannot tell dsDNA from RNA or free nucleotides, so a prep that is partly RNA reads high and the ratio drifts above 2.0.
- A microvolume instrument needs only 1–2 µL but has a 1 mm path, so it is a tenth as sensitive and much more dependent on a clean pedestal and a well-formed drop.
1What an absorbance reading actually is
A spectrophotometer shines a narrow band of light through your sample and compares what comes out with what went in. It reports absorbance, A = log₁₀(I₀/I), so A = 1 means a tenth of the light got through and A = 2 means a hundredth.
The blank
Run the same buffer the sample is in, in the same cuvette. Water is not elution buffer, and elution buffer is not an imidazole-containing eluate.
Path length
A 10 mm cuvette, a 2 mm micro-cuvette and a 1 mm microvolume pedestal give readings a factor of ten apart on the same sample.
Where to read
Below 0.05 the reading is noise and baseline drift; above about 1.8 stray light caps it and the instrument reads low.
Dilution
Dilute into the reading window and multiply the factor back. The dilution is the easiest part of the measurement to get right and the easiest to forget to record.
2Why 1.8 is a bad number to report
No monochromator is perfect. A small fraction of the light reaching the detector, usually a tenth of a percent to a few tenths, never passed through the sample at all: it scattered off optics, leaked around the slits, or came from a neighbouring wavelength.
3Reading purity off the shape of the spectrum
A single wavelength gives you a number with nothing to check it against. A full scan from 220 to 350 nm gives you the shape, and the shape is where the diagnosis lives.
- 230 nm
- Where chaotropic salts (guanidine), EDTA, phenol and carbohydrate absorb. A low A260/A230, under about 1.8, is the classic sign that a wash step was skipped, and it often appears while A260/A280 still looks perfect.
- 260 nm
- The nucleic-acid base peak. It counts bases, not molecules: dsDNA, ssDNA, RNA and free nucleotides all absorb here, with different conversion factors (50, 33 and 40 ng/µL per absorbance unit at 1 cm).
- 270 nm
- Phenol's peak. Phenol carry-over from an organic extraction shows as a shoulder on the long-wavelength side of the 260 nm peak, inflates the apparent yield, and drags A260/A230 down while leaving A260/A280 near enough to 1.8 to pass a quick glance.
- 280 nm
- The aromatic amino-acid band. Protein in a DNA prep pulls A260/A280 down towards 1.5; nucleic acid in a protein prep pushes A260/A280 up above 0.7 and inflates the protein concentration.
- 320–340 nm
- Nothing in a clean protein or nucleic-acid sample absorbs here, so any signal is particulates or aggregates scattering light. Use it as a background: if it is not close to zero, spin or filter the sample rather than subtracting your way out of trouble.
4When absorbance is not absorbance
An OD600 reading on a bacterial culture is not absorbance at all. Cells barely absorb at 600 nm; they scatter light out of the beam, and the instrument records the loss as though it were absorption.
Key terms
Practise these as flashcards →log₁₀ of the ratio of incident to transmitted light. Dimensionless, and defined only relative to the blank the instrument last measured.
A = ε·c·l. Absorbance is proportional to concentration and to path length, and contributions from different species add.
How strongly one species absorbs at one wavelength. Quoted molar (M⁻¹cm⁻¹) or as a specific absorbance for 1 mg/mL or 1% solutions.
The same buffer and cuvette as the sample, without the analyte. It sets I₀, so a wrong blank is a systematic error on every subsequent reading.
Light reaching the detector without passing through the sample. It puts a hard ceiling on absorbance and makes high readings come out low.
The distance the beam travels through the sample: 10 mm in a standard cuvette, about 1 mm on a microvolume pedestal, and whatever the liquid depth happens to be in a plate well.
Purity ratios for nucleic acids, about 1.8 and 2.0–2.2 for a clean prep. The first catches protein and the second catches chaotropes, phenol and EDTA.
The same number as absorbance, but used when the loss of light is mostly scattering rather than absorption, as in OD600 of a culture.
When to use it
- Quantifying a nucleic acid or protein prep in seconds, with a purity check thrown in, when you have at least a microlitre to spare and more than about 1 ng/µL of material.
- Tracking a bacterial or yeast culture's growth, deciding when to induce, and calculating a back-dilution or an inoculum.
- Following a reaction that changes colour or loses a chromophore (enzyme assays, nitrate and phosphate determinations, dye-binding protein assays) against a standard curve.
- Checking at a glance whether a column eluate contains anything at all, before committing it to a slower, more expensive assay.
Compared with alternatives
Dye-based fluorometric quantification is specific for double-stranded DNA and works a hundred-fold lower, so prefer it for dilute samples, for libraries, and whenever RNA might be present.
Bradford and BCA measure total protein against a standard curve and work on proteins with no tryptophan, but they are sensitive to detergents and reducing agents and consume sample.
A plate reader does the same absorbance measurement 96 times at once, but with a short and uncertain path length, so prefer a cuvette when the absolute number matters.
Counting cells on a haemocytometer or a counter gives you real cells per millilitre and viability, which OD600 cannot: turbidity cannot distinguish live cells, dead cells and debris.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Quantify DNA or RNA from the 260 nm base peak, using 50 ng/µL per absorbance unit for dsDNA, 40 for RNA and 33 for single-stranded DNA at a 1 cm path, and judge the prep from the A260/A280 and A260/A230 ratios.
- Blank on the elution buffer, not water: Tris, EDTA and carried-over salts all absorb below 240 nm and shift A260/A230.
- A260 cannot tell dsDNA from RNA or free nucleotides, so a prep that is partly RNA reads high and the ratio drifts above 2.0.
- A microvolume instrument needs only 1–2 µL but has a 1 mm path, so it is a tenth as sensitive and much more dependent on a clean pedestal and a well-formed drop.