Isolation of DNA

Hello everyone ,

DNA isolation is a process of purification of DNA from sample using a combination of physical and chemical methods. The first isolation of DNA was done in 1869 by Friedrich Miescher. Currently it is a routine procedure in molecular biology or forensic analyses.Purification or isolation of nucleic acids is the first step in most molecular biology studies and all recombinant  DNA techniques. Today I will be writing about Isolation of DNA from cell .

Source : the simplest source of DNA could be bacteria

The methods include

1) Cell lysis: It solubilizes Nucleic acid . This step include 

                      addition of SDS /Alkali

                       Boiling

                       addition of Chaotropic agents

2) Enzymatic treatment:Addition of protease (Protinase K), inhibit DNAse activity

                                      Addition of Nucleases, removes unwanted nucleic acid (RNAse or DNAse)

3) Phenol extraction:  Here equal volume volume of phenol is added to the sample and centrifuged. This allows partition of phase . As proteins are hydrophobic, thus partition in organic phase. Also DNA seen in aqueous phase in upper layer .

4) Ethanol precipitation: Here 0.3M sodium Acetate/ Potassium acetate / Ammonium acetate( pH 5-5.5) is added to neutralize the highly charged backbone and promote hydrophobic interactions.then equal volume of Ethanol /Isopropanol is added and incubated overnight at 4 °C .the precipitated DNA is collected by centrifugation . The pellet is rinsed with 70% Ethanol to remove excess of salt, dried and dissolved in buffer .

5)Adsorption Methods: This step mainly separates RNA from sample.

The above sample as obtained above is mixed with a resin ( silica or diatomaceous earth). the DNA binds to the matrix but RNA do not. the DNA is eluted with low salt buffer.

ThankYou

with love 

-Dixy