Isolating Bacterial Genomic DNA

Isolation and Purification of Genomic DNA from Bacteria

The general procedure for purification of genomic bacteria requires several chemicals, enzymes and reagents which are enlisted as follows:

1. Tris EDTA (TE) Buffer: It consists of Tris HCl and Tris HCl helps in maintaining the pH, while EDTA is a chelating agent which restricts the activity of nucleases and several other enzymes.
2. Lysozyme: Lysozyme is enzyme used to degrade the cellular components.
3. SDS (Sodium dodecyl sulfate): SDS is a detergent which lyses the bacterial cell walls.
4. Proteinase K: Proteinase K is the enzyme used to degrade the cellular protein molecules.
5. CTAB (Hexadecyl trimethyl ammonium bromide): CTAB forms complex with denatured protein, cell wall debris, and polysaccharides in form of precipitates.
6. NaCl (Sodium Chloride)
7. Chloroform: Isoamyl Alcohol (24:1): It binds to the proteins and lipids causing them to precipitate out. This is because the lipids and proteins are non aqueous compounds while DNA is aqueous.
8. Isopropanol: It is added for the precipitation of proteins.
9. 70 % Ethanol: It helps in the precipitation of DNA. 

THE BASIC PRINCIPLE

The basic principal behind the extraction of DNA from any organism can be understood in very simple way. We all know that the genetic material of bacteria is present inside the cytoplasm. This means the first objective must be to break open the bacterial cell. This can be done by partially or completely destroying the cell wall. Now when the cell wall has been destroyed the genetic material can be taken out. But there is just one difficulty. The cytoplasm contains a hell lot of different molecules. These molecules will definitely mix up with the genetic material thereby degrading its quality. Please note that experiments involving genetic materials need to be highly accurate and precise. This can only happen if first of all we are able to isolate pure DNA. So what should be the next step? What do we need to do to separate out the DNA leaving behind rest? Here comes the time when we need to use different solutions and chemicals along with the centrifuge machine to separate out what we want. The entire process has been described in the following sections.

 

 

STEPS INVOLVED:

1. The bacterial cells are cultured in LB Broth. The cells are the transferred to a centrifuge tube and centrifuged at 10,000 rpm for 2 minutes.
2. The supernatant is decanted and the pellet which contains the bacterial cell is resuspended in TE Buffer. SDS is added and the suspension is incubated at 37° C for about one hour. The mixture of chloroform: Isoamyl alcohol is added and the centrifuge tube is centrifuged at 12,000 rpm for ten minutes.
3. The aqueous phase is transferred to a new tube and CTAB and chilled Isopropanol is added. A small amount of lysozyme is added and the centrifuge tube is centrifuged at 12,000 rpm for ten minutes.
4. The supernatant is discarded and washed with 70 % ethanol. A small amount of RNase is added and the tube is again centrifuged at 12,000 rpm for 10 minutes.
5. The supernatant is discarded and washed with 70% ethanol. It is then air dried and resuspended in TE Buffer. The DNA isolation is thus complete.

The isolation of pure genomic DNA from bacteria can be done easily by following the above mentioned steps. I hope the so much complicated procedure would now look a bit simpler to you. One must remember that there are various protocols followed by different scientists all over the world. The chemicals used may be different but the basic idea behind each of the procedure is the same. Culture the bacteria, break open the cell and isolate the DNA. Believe me the method explained by me is just enough for the simple laboratory requirements. But if you are a dedicated researcher, you are recommended to follow any of the accepted protocols that suit your need.

For a more detailed tutorial please watch the video. The link for the video will be updated soon.

For notes and explanation booklet please download the pdf format booklet from the website www.regreenindia.yolasite.com.