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6 Feb 2010 10:35:53 IST

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How to isolate DNA

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How to isolate DNA

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edison

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29 Jan 2012 18:47:57 IST

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DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic and two optional steps in a DNA extraction:

Breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by chemical and physical methods-blending, grinding or sonicating the sample.
Removing membrane lipids by adding a detergent or surfactants.
Removing proteins by adding a protease (optional but almost always done).
Removing RNA by adding an RNase (often done).
Precipitating the DNA with an alcohol — usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt.

Refinements of the technique include adding a chelating agent to sequester divalent cations such as Mg²⁺ and Ca²⁺, which prevents enzymes like DNase from degrading the DNA.

Cellular and histone proteins bound to the DNA can be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation.

If desired, the DNA can be resolubilized in a slightly alkaline buffer or in ultra-pure water.

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