Isolation of Genomic DNA from bacteria: Phenol- Chloroform method

Isolation of Genomic DNA from bacteria is a little tedious and time-consuming methods but a useful and crucial method for the isolation of bacteria from soil and helps in characterizing the soil bacteria. As we know that DNA is the main part of life and can be so useful for the identification of organisms’ nucleotides.

First, the important part is to isolate pure culture from the bacterial colonies by a different method that we discussed in our previous article, then only we can isolate Genomic DNA from the bacteria by different methods. We are going to discuss the Phenol chloroform method for this process. A variety of chemicals and enzymes were used in this specific method. We will be going to explain all the functions of those chemicals too.

Isolation of Genomic DNA from bacteria principle

The fundamental method of isolating DNA involves breaking down the cell wall, nuclear membrane, and cell membrane to release the highly intact DNA into solution. Next, DNA is precipitated while contaminating biomolecules like proteins, polysaccharides, lipids, phenols, and other secondary metabolites are removed.

Isolation of Genomic DNA from bacteria protocol:

Genomic DNA was isolated from bacteria by Phenol and chloroform method.

1. First of all, we have to isolate a pure culture of bacteria by different methods, and then only after seed culture, a small part will be taken for fermentation for extracting secondary metabolites.
2. And another part of the seed culture about 15mL of pure cultures was taken in a falcon tube.
3. After centrifugation at about 5 minutes at 8000 rpm and the supernatant was removed.
4. Cell pellets were collected and the process of isolation of genomic DNA was started.
5. Harvested cell pellets were then lysis with lysis buffer that is 10% sucrose and was vertex strongly followed by centrifugation and this process was repeated 2 times.
6. The supernatant was removed and the pellets were mixed with 240 microliters of 8mg/mL lysozyme and the sample was kept at 37 degrees Celcius.
7. After complete lysis, 240 microlitres of 0.5M EDTA and 26 microlitres of 10 mg/mL proteinase K were added.
8. The samples were again kept in a water bath for 5 minutes at 37 degrees Celcius.
9. SDS (10%) 200 microlitres was added and samples were kept at 70 degrees Celcius for 10 minutes and were immediately transferred to an ice bath for about 15 minutes.
10. Then an equal volume of Phenol: Chloroform: isoamyl alcohol reagent (25:24:1) was added to the samples and was shaken gently, and centrifuged at 8000 rpm for 5 minutes.
11. Now the three layers were observed, the upper layer was aqueous in which cell DNA was present, the middle interphase, and the lower one is an organic layer. The supernatant was transferred into a new Eppendorf tube without disturbing the solvent layer.
12. Then, 40 microlitres of 1mg/mL RNase was added and kept at 37 degrees Celcius for an hour.
13. After all these enzymes and chemicals, 0.8 volumes of isopropanol and 2 volumes of 100 % alcohol were added into the tube and shaken slowly.
14. For the precipitation of DNA, the tubes were incubated at -20 degrees Celcius for 30 minutes.
15. After this process, the solution was centrifuged and washed with 70% ethanol and the remaining ethanol was evaporated by incubation at room temperature overnight.
16. Finally, after putting it overnight, 10 microlitres of tris-base-EDTA (TE) buffer was added to the tubes and we get the DNA after centrifugation at 5000rpm for 10 seconds.
17. Now, the genomic DNA was examined through Gel electrophoresis.

Chemicals used during isolation and their function:

The various chemicals used during the isolation of genomic DNA are:

1. Tris-buffer- It generally maintains the Ph of the solution and helps in making the membrane permeable resulting in lysis.
2. EDTA- A chelating agent, chelates the magnesium ions which act as a cofactor for the enzyme.
3. SDS- Act as detergents, which helps in the lysis by removing lipid molecules and thereby causing disruption of cell membranes.
4. Acetate- Protect from denaturation
5. Phenol- The addition of phenol breaks the bond between amino acids leading to the denaturation of protein.
6. Chloroform- Allow proper separation of the organic and aqueous phases.
7. Isoamyl alcohol- It prevents the emulsification of the solution.

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