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Isolation of genomic DNA from Escherichia coli by the phenol-chloroform method

Aim: To isolate and extract high quality genomic DNA from a 24 hour old culture of Escherichia coli using the liquid-liquid phenol chloroform extraction method.

Theory:

Genomic DNA is the complete set of DNA found within an organism's genome, encoding all the genetic information required for its development, function, and reproduction. In prokaryotes like Escherichia coli (E. coli), the genomic DNA is a single, circular, double-stranded molecule (circular dsDNA molecule) located in the cytoplasm. Unlike eukaryotes, prokaryotes lack a nucleus, and their DNA is compacted within a region called the nucleoid. The DNA molecule is associated with various proteins that aid in its supercoiling and packaging, making its extraction a precise and delicate process.

For isolation of genomic DNA of E. coli, we need to disrupt the cell and then isolate DNA from it. For destroying the cell wall of bacteria, we need and lysozyme which act on peptidoglycan, then the cell membrane is disrupt using lysis buffer which contain SDS. To remove proteins and protect the DNA, Proteinase K is added, the mixture of phenol-chloroform-isoamyl alcohol is used to dissolve lipids and denature proteins, leading to the formation of distinct layers in the solution after centrifugation. The aqueous layer contains the DNA which is precipitated from the aqueous phase by adding a precipitation agent such as isopropanol or chilled ethanol. These agents cause DNA molecules to come out of solution and form a visible white pellet upon centrifugation. Now the DNA is extracted from this precipitate.

The final step involves resuspending the purified DNA pellet in a buffer solution like TE buffer (Tris-EDTA) to stabilize and store the DNA. TE buffer maintains the pH and ionic strength necessary to prevent DNA degradation and facilitate downstream molecular biology applications, such as PCR (Polymerase Chain Reaction) or sequencing.

Requirements:

COMPONENTS	USES
E. coli overnight culture in LB medium	Genomic DNA isolation
TE Buffer (pH 8)	Maintains pH and chelate divalent ions, protect DNA
Lysis Buffer	Breaks open cells and releases DNA
Lysozyme (1 mg/ml)	Digests peptidoglycan layer in E. coli cell wall
Proteinase K (20 mg/ml)	Digests proteins and protects DNA from DNases
Phenol: Chloroform: Isoamyl Alcohol (25:24:1)	Purifies DNA by separating proteins
Ethanol (100%, chilled)	Precipitates DNA for easy collection
Ethanol (70%)	Washes DNA to remove impurities and salts
Sodium Acetate (3M, pH 5)	Enhances DNA precipitation in alcohol
Eppendorf tubes, Micropipette, Tips, Centrifuge, Laminar air flow etc.

 

Preparation of chemicals:

·       TE Buffer: (10 mM Tris-HCl, 1 mM EDTA, pH 8)

Dissolve 1.21 g of Tris-HCl and 0.292 g of EDTA in distilled water, adjust pH to 8.0, and make up to 1 L.

·       Lysis Buffer: (10 mM Tris-HCl, 100 mM EDTA, 10% SDS)

Dissolve 1.21 g of Tris-HCl, 37.2 g of EDTA, and 5 g of SDS in deionized water, adjust pH to 8.0, and make up to 1 L.

·       Phenol: Chloroform: Isoamyl alcohol: (25:24:1)

Mix 25 parts phenol, 24 parts chloroform, and 1part isoamyl alcohol

   - Phenol denatures and removes proteins.
   - Chloroform enhances phase separation.
   - Isoamyl Alcohol prevents foaming, ensuring efficient handling.

·       Sodium Acetate: (3M, pH 5)

Dissolve 24.6 g in 80 ml distilled water, adjust pH to 5 by adding glacial acetic acid, and make up to 100 ml.

 

Procedure:

Step 1: Cell Harvesting

1. Grow E. coli overnight in LB medium at 37°C.
2. Centrifuge 3 ml of culture at 10,000 rpm for 5 minutes to pellet cells.
3. Discard the supernatant, then resuspend the pellet in 500 µl of TE buffer.

Step 2: Cell Lysis

1. Add 10 µl of lysozyme to the resuspended cells to digest the cell wall.
2. Incubate at 30°C for 1 hour.
3. Add 100 µl of lysis buffer to the cell suspension.
   - Tris-HCl in the lysis buffer maintains a stable pH.
   - EDTA chelates Mg²⁺ ions, inhibiting DNases that could degrade DNA.
   - SDS is a detergent that disrupts the cell membrane, releasing DNA.
4. Add 50 µl of Proteinase K (20 mg/ml) and mix thoroughly.
5. Incubate the mixture at 37°C for 1 to 2 hours to fully lyse cells and release DNA.

Step 3: DNA Extraction

1. Add 400 µl of Phenol: Chloroform: Isoamyl alcohol (25:24:1) and gently mix.
2. Centrifuge at 10,000 rpm for 10 minutes to separate the phases.
3. Carefully transfer the aqueous (top) layer (which contains DNA) to a new tube.

Step 4: DNA Precipitation

1. Add 1/10 volume of 3M sodium acetate to the DNA solution, followed by 2 volumes of 100% chilled ethanol, and gently mix by inverting.
2. Centrifuge at 10,000 rpm for 10 minutes to pellet the DNA.

Step 5: DNA Washing

1. Discard the supernatant and add 500 µl of 70% ethanol to the DNA pellet.
2. Centrifuge at 10,000 rpm for 5 minutes, discard the ethanol and air-dry pellet.

Step 6: DNA Resuspension

1. Resuspend the DNA pellet in 50µl of TE buffer.
   - Store the purified DNA at -20°C for long-term storage.

Observation: After adding chilled ethanol, We see the white, stringy precipitates floating in the tube. The solution becomes cloudy at first and then the DNA fibers start to clump together like a small cotton ball.

Result: The genomic DNA was successfully isolated from E. coli as confirmed by the presence of a sharp band on 0.8 percent agarose gel electrophoresis.

Conclusion: The extraction process worked well because we avoided harsh mixing. Using chilled chemicals is the secret for getting a good pellet. This DNA can now be used for further studies like PCR or restriction digestion.

Note: This protocol is for educational use in standard teaching laboratories. Small variations may occur depending on lab conditions and handling. Results may vary. biologynotes.in cannot take responsibility for any errors, misuse, or results obtained. Always follow proper lab safety and instructor guidance.