How to Calculate DNA Concentration From A260

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.

The copy number worked out

Copies = mass in grams × 6.022 × 10²³ ÷ (length in bp × 618). For 1 ng of a 3,000 bp plasmid: 1 × 10⁻⁹ × 6.022 × 10²³ ÷ (3,000 × 618) = 6.022 × 10¹⁴ ÷ 1,854,000 = 3.25 × 10⁸ copies. Some protocols use 650 g/mol per base pair, which gives about 5% fewer.

Pipetting a set amount

To put 100 ng into a reaction from a 125 ng/µL stock you need 100 ÷ 125 = 0.8 µL, which is too small to pipette accurately. Dilute the stock 1:10 to 12.5 ng/µL first and add 8 µL instead.

Common problems

• The DNA reading is too high. RNA absorbs at 260 nm too, so a DNA prep with RNA in it reads high. Treat with RNase, or use a dye-based assay that sees only DNA.
• The 260/280 ratio is low for clean DNA. Unbuffered water is slightly acidic and can lower the ratio by 0.2 to 0.3. Dilute and blank in a buffer such as TE.
• Readings jump between drops. Mix the sample before measuring, wipe both pedestals, and watch for bubbles in microvolume instruments.
• A negative or tiny reading. The blank used a different buffer from the sample, or the sample is below the instrument's useful range.