Isolation of DNA

Isolation of DNA

Aim

To isolate the hereditary element (DNA) from Escherichia coli.

Principle

Bacterial genetic material is high molecular weight double stranded, highly supercoiled circular DNA.  The isolation of DNA is a very important step in various procedures in molecular biology, in many areas of bacterial physiology, genetics and biochemistry. Purified DNA is required for many applications such as studying DNA structure and chemistry, examining DNA-protein interactions, carrying out DNA hyrbridizations, sequencing or PCR, performing various genetic studies or gene cloning.  Several methods are used for the isolation of genomic DNA from various types of cells or tissues.  Generally, all methods involve the disruption and lysis of cells. This is followed sometimes by the removal of RNA (by RNAses, salt or other methods).   DNA is separated from proteins by several methods including digestion of proteins by the enzyme proteinase K or by salting-out, organic extraction, or binding of the DNA to a solid-phase support (such as an anion-exchange column or silica technology). DNA is finally recovered by ethanol precipitation or isopropanol precipitation.

In general, the separation of DNA from cells and cellular components can be divided into four stages:

1.Cell disruption

2.Lysis of Cell

3.Removal of Proteins and other contaminants

4.Recovery of DNA

In some genomic DNA isolation protocols, stages 1 and 2 are combined.

SDS (sodium dodecyl sulfate) is usually used to lyse the cells.  It is an anionic detergent that disrupts the lipid layer, helps to dissolve membranes and binds positively charged chromosomal proteins to release the DNA into solution.  DNA is stabilized and remains as a double helix in the presence of sodium chloride.  EDTA (Ethylene Diamine Tetra Acetic acid), a chelating agent, chelated Mg²⁺, which is a cofactor of DNAse that chews up DNA rapidly.  Organic solvent extraction using Phenol: Chloroform: Isoamyl alcohol (25:24:1) is done to remove protein from DNA because hydrophobic residues in the protein interact with organic solvents.  pH of the buffer should be 8 or above because at low pH, DNA will not be in aqueous phase.  Purified DNA is then dissolved in ethanol.

Materials required

1. Overnight culture of E coli 

2. Centrifuge

3. SET buffer (150 mM NaCl, 1 mM EDTA, 10 mM Tris, pH- 8.3)

4. 10% SDS

5. Water bath at 37^oC

6. Phenol : chloroform : isoamyl alcohol (25:24:1)

7. 7 M ammonium acetate

8. Isopropanol

9. 70% ethanol

Procedure

1. 10 ml of overnight culture of E coli in nutrient broth was centrifuged at 4000 rpm for 10 minutes.

2. Supernatant was discarded and pellet was resuspended in 1 ml of SET buffer.

3. To the above suspension, 100 µl of 10% SDS was added for cell lysis, and incubated in a water bath at 37^oC for 30 minutes.

4. After 30 minutes, phenol-chloroform extraction was done by vigorous shaking to remove debris protein by adding 1 ml of Phenol: Chloroform: Isoamyl alcohol mixture and subsequently centrifuged at 4000 rpm for 10 minutes.

5. To the final aqueous phase separated from the extraction mixture, a mixture of 0.5 ml of 7 M ammonium acetate and 1.2 ml of Isopropanol was added.

6. The precipitate was washed with 70% ethanol and dissolved in 1X TE buffer.

Observation

During the addition of Ammonium acetate and Isopropanol to the supernatant, the DNA was precipitated.  The precipitated DNA was seen like a cluster of threads in a suspension.

Result

The genomic DNA was isolated from Escherichia coli.