From absorbance to concentration
Nucleic acids absorb ultraviolet light at 260 nm. With a 1 cm path, an A260 of 1.0 corresponds to about:
- 50 ng/µL of double-stranded DNA
- 40 ng/µL of RNA
- 33 ng/µL of single-stranded DNA
Concentration = A260 × factor × dilution. A sample diluted 1:10 that reads 0.25 holds 0.25 × 50 × 10 = 125 ng/µL of dsDNA. Readings are most reliable between about 0.1 and 1.0; dilute stronger samples. Short oligos vary more with their base composition, so they are better calculated from their own extinction coefficient.
Purity ratios
- A260/280 around 1.8 for DNA and 2.0 for RNA. Lower values suggest protein or phenol.
- A260/230 around 2.0 to 2.2. Lower values suggest guanidine salts, EDTA or carbohydrates left over from the prep.
From ng to copies
For qPCR standards you need the number of molecules. One base pair of dsDNA weighs about 618 g/mol, so 1 ng of a 3,000 bp plasmid is about 3.2 × 108 copies.
Calculators
The DNA Concentration Calculator takes your readings and the dilution and checks the ratios. The DNA Copy Number Calculator converts between ng and copies. For other molecules, the Beer-Lambert Law Calculator works from the extinction coefficient.