Analysing DNA

Spectrophotometric measurement of DNA concentration

The concentration of DNA and RNA should be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer. For accuracy, absorbance readings at 260 nm should fall between 0.15 and 1.0.

Pure DNA has an A260/A280 ratio of 1.8–2.0 in 10 mM Tris·Cl, pH 8.5.

Strong absorbance at A280 resulting in a low A260/A280 ratio indicates the presence of contaminants, such as proteins.

Strong absorbance at 270 nm and 275 nm may indicate the presence of contaminating phenol.

Absorbance at 325 nm suggests contamination by particulates in the solution or dirty cuvettes.

Spectrophotometric conversions from absorbance at 260 nm
1 A260 unit Concentration (µg/ml)*
dsDNA 50
ssDNA 33
Oligonucleotides 20–30