Purification of DNA | Molecular Biology

In this article we will discuss about the principle, requirements and procedure for purification of DNA.

Principle:

DNA is removed from gel by dissolving the gel in cesium chloride and DNA is taken out with ethidium bromide in T.E. buffer, centrifuged and DNA is removed. Ethidium bro­mide is extracted with n-butanol, and isopropanol precipitates out DNA.

Requirements:

Reagents:

Procedure:

1. To 1 ml of DNA in T.E. buffer add 0.75 g of Cesium chloride and dissolve this completely.

2. Add 10 m/ml ethidium bromide in T.E. buffer and transfer this to an ultra- centrifuge tube and centrifuge at 12,000 ‘ rpm for 15-20 min at 15°C.

3. Collect DNA with the aid of a Pasteur pipette viewing in a U.V. transilluminator.

4. Using water-saturated n-butanol, extract out ethidium bromide till the extract be­comes colourless.

5. Add an equal volume of isopropanol and keep for 1 hour at -20°C to precipitate DNA.

6. Centrifuge, wash the pellet with ice cold 80% ethanol, dry and resuspend the DNA pellet in small volume of T.E. buffer.

7. Store aqueous DNA preparation frozen at -70°C. For longer duration store un­der ethanol.

The spectral characteristics of good DNA preparations are:

A ₂₃₀ = <0.1 A₂₃₀/ A₂₆₀ = 0.45 A₂₈₀/A₂₆₀ = < 0.55

DNA extinction coefficient is E1%^1cm = 200 or

DNA at a concentration of 50 μg/ml shows an absorbance of 1 at 260 nm.