DNA Concentration Calculator
Enter an absorbance reading at 260 nm to get the concentration of your DNA or RNA sample in µg/mL, ng/µL, and nM. The calculator also converts between mass concentration and molarity using fragment length, generates a C1V1 dilution protocol, and evaluates A260/A280 and A260/A230 purity ratios with automatic quality alerts. Choose dsDNA, ssDNA, RNA, or oligonucleotide to apply the correct extinction factor.
How UV absorbance is used to measure DNA concentration
Nucleic acids absorb ultraviolet light most strongly at 260 nm because the aromatic rings of the nucleotide bases are chromophores. The relationship between absorbance and concentration follows Beer-Lambert law: C = A260 / (path length x extinction coefficient), or equivalently, C = A260 x CF x dilution factor, where CF is a convenient empirical conversion factor that bundles the molar extinction coefficient with the average molecular weight per base. For double-stranded DNA the CF is 50 µg/mL per absorbance unit at 1 cm path length; for single-stranded DNA and oligonucleotides it is 33, and for RNA it is 40, reflecting the differing molecular weights and stacking interactions between the base types.
Interpreting A260/A280 and A260/A230 purity ratios
Proteins absorb strongly at 280 nm (due to tryptophan and tyrosine residues), so the ratio A260/A280 is used as an index of protein contamination. Pure double-stranded DNA gives a ratio between 1.7 and 2.0; pure RNA gives 1.8 to 2.1. A low ratio suggests residual protein or phenol; a high ratio can indicate RNA carry-over or UV-absorbing contaminants. The ratio A260/A230 reflects contamination by organic solvents and chaotropic salts - phenol, guanidinium thiocyanate, EDTA, and polysaccharides all absorb around 230 nm. An acceptable A260/A230 lies between 1.8 and 2.2. Ratios below this threshold strongly suggest that additional purification (a silica column clean-up or precipitation step) is needed before the sample can be used for sensitive downstream workflows such as next-generation sequencing or quantitative PCR.
Converting between mass concentration and molarity
Mass concentration (µg/mL or ng/µL) tells you how much DNA is present by weight, but many molecular biology applications require a molar concentration - particularly ligation reactions, where a 3:1 insert-to-vector molar ratio is needed, or library preparation for next-generation sequencing, where inputs are specified in nM or fmol. To convert, you need the average molecular weight of the fragment, which is approximately 650 g/mol per base pair for dsDNA, 330 g/mol per base for ssDNA, and 340 g/mol per base for RNA. Dividing the mass concentration (in µg/mL) by the molar mass (in g/mol) and multiplying by 10^6 converts to nM. The number of molecules per microlitre follows from the molar concentration multiplied by Avogadros number (6.022 x 10^23) and appropriate unit conversions.
Setting up a dilution using C1V1 = C2V2
When a measured stock is too concentrated for a downstream reaction, the dilution equation C1V1 = C2V2 gives the volumes to combine. C1 is the stock concentration, V1 is the volume of stock to take, C2 is the target concentration, and V2 is the final volume you need. Rearranging: V1 = (C2 x V2) / C1. The remaining volume (V2 minus V1) is filled with nuclease-free water or TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). TE is preferred for long-term storage because the EDTA chelates divalent cations that activate DNase enzymes; nuclease-free water is used when the EDTA would interfere with downstream enzymes such as polymerases.
Conversion factors and purity ratio reference
| Sample type | CF (µg/mL per A260) | MW per unit (g/mol) | A260/A280 target | A260/A230 target |
|---|---|---|---|---|
| dsDNA | 50 | 650 (per bp) | 1.7-2.0 | 1.8-2.2 |
| ssDNA | 33 | 330 (per base) | 1.7-2.0 | 1.8-2.2 |
| RNA | 40 | 340 (per base) | 1.8-2.1 | 1.8-2.2 |
| Oligonucleotide | 33 | 330 (per base) | 1.7-2.0 | 1.8-2.2 |
Standard extinction coefficients for nucleic acid quantification by UV spectrophotometry at 260 nm (1 cm path, dilution factor 1).
Frequently asked questions
What is the conversion factor (CF) for DNA at 260 nm?
The empirical conversion factor is 50 µg/mL per absorbance unit for double-stranded DNA (dsDNA), 33 for single-stranded DNA and oligonucleotides, and 40 for RNA. These values assume a 1 cm path length and an undiluted sample. They come from dividing the average molar extinction coefficient by the average molecular weight per base (pair), and are widely cited in Sambrook and Russell, Molecular Cloning.
What is a good A260/A280 ratio for DNA?
For pure double-stranded DNA, the A260/A280 ratio should be between 1.7 and 2.0, with 1.8 considered ideal. A ratio below 1.7 typically signals protein contamination, residual phenol from extraction, or RNA degradation. A ratio above 2.0 can indicate RNA carry-over. RNA samples should show a ratio between 1.8 and 2.1.
Why is my A260/A230 ratio low?
A low A260/A230 ratio (below 1.8) usually means the sample contains residual chaotropic salts (guanidinium thiocyanate or guanidinium hydrochloride from column-based extraction kits), phenol, EDTA, or polysaccharides, all of which absorb around 230 nm. Remedies include repeating the wash steps on the silica column, performing an ethanol precipitation, or using a commercial clean-up column. A ratio below 1.5 often renders the sample unsuitable for sensitive applications like qPCR or library prep without additional purification.
How do I convert ng/µL to nM for a DNA fragment?
Divide the mass concentration in ng/µL (equivalent to µg/mL) by the molecular weight of the fragment in g/mol, then multiply by 10^6. For dsDNA, molecular weight in g/mol is approximately 650 times the number of base pairs (e.g. a 500 bp fragment has MW approximately 325,000 g/mol). A 25 ng/µL solution of a 500 bp fragment is therefore (25 / 325,000) x 10^6 = 76.9 nM.
What path length does a NanoDrop use?
The Thermo Scientific NanoDrop uses a very short path length, nominally 1 mm (0.1 cm) for concentrated samples and 0.5 mm for highly concentrated ones, but it corrects the reading internally and reports the equivalent 1 cm absorbance value. When you enter a reading from a NanoDrop into this calculator, use a path length of 1 cm because the instrument has already normalized the measurement to that standard path length.
What is the difference between µg/mL and ng/µL?
They are numerically identical. 1 µg/mL = 1 ng/µL, because 1 µg/mL = 1 µg per 1000 µL = 0.001 µg/µL = 1 ng/µL. The two units are used interchangeably in the molecular biology literature, but ng/µL is more common when reporting small volumes and µg/mL is used in larger-scale contexts. This calculator displays both.
How do I calculate the volume of stock DNA to use in a dilution?
Use the dilution equation C1V1 = C2V2, where C1 is your stock concentration, V1 is the volume of stock to take, C2 is the target concentration, and V2 is the final volume you need. Rearrange to V1 = (C2 x V2) / C1. For example, to prepare 50 µL at 10 ng/µL from a 100 ng/µL stock: V1 = (10 x 50) / 100 = 5 µL of stock, then add 45 µL of water or buffer.