DNA and RNA quantification
Concentration and purity from A260, with the conversion factor made explicit.
Genomic DNA, plasmid preps, PCR products — anything duplex.
Exactly what the instrument displayed.
A cuvette is 1 cm. A microvolume instrument uses a far shorter path and usually reports the 1 cm equivalent already — if yours shows the raw reading, put the real path here.
Gives the total yield.
Concentration
21 µg/mL · 0.021 mg/mL
Total yield
A260 / A280
~1.8 for clean double-stranded dna
A260 / A230
~2.0 to 2.2 for a clean prep
c = A₂₆₀ ÷ path × factor × dilution- Purity ratios and what shifts them — BioTechniques, 1997
Absorbance cannot tell nucleic acid from free nucleotides, and it counts DNA in an RNA prep as though it were RNA. Where the distinction matters, a fluorescent assay is specific and stays accurate far lower. The 260/280 ratio also shifts with pH, so read against the same buffer the sample is in.
When to use this
Use this to turn a spectrophotometer reading into a DNA or RNA concentration, and to judge whether the prep is clean enough to use. Absorbance cannot distinguish nucleic acid from free nucleotides, so for low or contaminated samples a fluorescent assay is the better measurement.
Worked example
A plasmid prep read on a cuvette spectrophotometer.
- Sample is
- Double-stranded DNA
- A260
- 0.42
- A280 / A230
- 0.23 / 0.19
- Path length
- 1 cm
Result
Ratios of 1.83 and 2.21 are both healthy, so the number can be trusted for a downstream reaction.
What people get wrong
- Using the dsDNA factor for RNA. The conversion is 50 µg/mL per A260 for duplex DNA and 40 for RNA, so the wrong one overstates an RNA prep by a quarter.
- Reading above about 1.0 absorbance. Past that the detector under-reports without complaining; dilute and read again.
- Ignoring a low 260/230. Guanidine and phenol carried through an extraction inhibit downstream enzymes even when the concentration looks perfectly good.
Questions
+What should the ratios be?
About 1.8 at 260/280 for DNA and 2.0 for RNA, and roughly 2.0 to 2.2 at 260/230 for both. Lower at 280 suggests protein; lower at 230 suggests salts, phenol or carbohydrate.
+My microvolume instrument reports ng/µL already — do I need this?
For the concentration, usually not. For the interpretation, yes: this states which factor was applied and what the ratios imply, which the instrument does not.
+Why does the 260/280 ratio change between readings?
It is sensitive to pH and ionic strength. Read against the same buffer the sample is in, and expect a slightly lower ratio in pure water.
Related tools
- Dilution calculator — Work out how much stock and diluent to combine for a target concentration.
- Molarity calculator — Convert between mass, molar concentration and volume for a solution.
- Peptide analyser — Mass, isoelectric point, extinction coefficient and composition from a sequence.
Science last reviewed .