Characterization by spectrophotometric method.
The DNA isolated from living cells is usually contaminated with protein, RNA,
and salts used during the isolation process. The purity of DNA may be estimated
by utilizing the property of the heterocyclic rings of the nucleotides of absorbing
light strongly in the UV range. DNA absorbs maximum light energy at about
260 nm. An optical density of 1.0 corresponds to approximately 50 µg/mL of
double stranded DNA. The ratio of absorbance viz. A260/A2SO and A2SO/
A260 provides an estimation regarding the purity of DNA. A typically pure
preparation of good-quality DNA should exhibit the following spectral properties:
- A260/A2SO ≈ 1.80
- A2SO/A260 ≈ 0.55
- Sample DNA
- TE buffer:Tris-HCI, 10 mM EDTA, 1 mM; pH 8.0
- Spectrophotometer and quartz cuvette
To find out the purity of DNA, make the appropriate dilution with TE buffer,
and measure the absorbance at 260 nm and 280 nm.
Do not use glass or plastic cuvettes, as lights in the UV range do not pass
- Calculate A260/A280 and A280/A260, and check if the values are within
the acceptable limit.
- Calculate the Dt~A concentration as follows:
Concentration of DNA = (A260 × 50 × dilution factor) in mg/mL.