DNA & Protein Lab

How much DNA or RNA is in your tube, and how clean is it? From a spectrophotometer reading at 260 nm get the concentration, the total yield and the purity ratios A260/A280 and A260/A230. Add the length for the molarity, the picomoles and the number of copies, or start from a known concentration to convert nanograms into moles.

Molecular biologySpectrophotometryNucleic acids

Inputs

Input examples

One absorbance unit at 260 nm (1 cm light path) is about 50 µg/mL of double-stranded DNA, 37 µg/mL of single-stranded DNA and 40 µg/mL of RNA. For oligos the factor depends on the sequence: use the Primers & oligos tab, or enter your own factor. Enter the absorbance of the measured solution and how much you diluted the sample before measuring. Microvolume instruments (NanoDrop and similar) already report values for a 10 mm path. The purity ratios and the length are optional. 1 ng/µL = 1 µg/mL.

Sample

Start from

A260

Dilution factor — 1 if undiluted

A280 — optional

A230 — optional

Light path — mm (10 for a standard cuvette)

Sample volume — µL, optional

Length — bp, optional

Measuring DNA With Light

The bases of DNA and RNA absorb ultraviolet light most strongly at 260 nm, so the absorbance at that wavelength measures how much nucleic acid there is (the Beer–Lambert law). Single strands absorb more than double strands, because stacked bases in a helix shield each other: the same absorbance means less RNA or ssDNA than dsDNA.

Proteins absorb most at 280 nm and many contaminants (phenol, guanidine salts, carbohydrates) at 230 nm. Pure DNA has an A260/A280 of about 1.8 and pure RNA about 2.0; lower values point to protein or phenol. An A260/A230 of 2.0–2.2 is clean; lower values point to salts or other leftovers from the purification.

For cloning and PCR you often need moles rather than grams: how many copies of a plasmid, or a 3 : 1 ratio of insert to vector. Dividing the mass by the molecular weight, about 650 g/mol per base pair, gives the moles; multiplying by Avogadro’s number gives the copies.

A260 = 1≈ 50 µg/mL dsDNA

260/280≈ 1.8 DNA, 2.0 RNA

260/2302.0–2.2 when clean

reliable rangeA260 between about 0.1 and 1

Purity at a Glance

ratios

Result

Enter the reading above

Worked Techniques

Absorbance to Concentration

Multiply the reading by the factor for the kind of nucleic acid and by the dilution. For a light path other than 1 cm, divide by the path in cm.

Example: in 50 µL that is 18.75 µg of DNA.

Mass to Moles

A plasmid of 3000 bp weighs 1.82 million g/mol. Divide the concentration in g/L by this for mol/L.

Example: 1 µg of this plasmid is 0.549 pmol, or 3.3 × 10¹¹ copies.

Copies per Microlitre

Used to make dilutions for qPCR standards or to count genome copies.

Example: 375 ng/µL of the 3 kb plasmid: 1.24 × 10¹¹ copies per µL.

Sources & Verification

References

DNA and RNA Molecular Weights and Conversions, Thermo Fisher Scientific (Ambion technical resources), read 5 November 2026 — A260 factors (dsDNA 50, ssDNA 37, ssRNA 40 µg/mL) and the approximate molecular weights from the length.
M. R. Green and J. Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, 2012 — quantifying nucleic acids by absorbance and the purity ratios.
BIPM, The International System of Units, 9th ed., 2019 — N_A = 6.022 140 76 × 10²³ mol⁻¹.