Estimation of DNA purity and Quantification

Characterization by spectrophotometric method.

Introduction
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

Materials

• Sample DNA
• TE buffer:Tris-HCI, 10 mM EDTA, 1 mM; pH 8.0
• Spectrophotometer and quartz cuvette

Procedure
To find out the purity of DNA, make the appropriate dilution with TE buffer, and measure the absorbance at 260 nm and 280 nm.  

Notes
Do not use glass or plastic cuvettes, as lights in the UV range do not pass through these.

Observations

1. Calculate A260/A280 and A280/A260, and check if the values are within the acceptable limit.
2. Calculate the Dt~A concentration as follows:
   Concentration of DNA = (A260 × 50 × dilution factor) in mg/mL.