Simple Autoclave Diagram

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.

Principle: Bacterial cells are small but packed tightly with DNA. So first we need to break them open properly. SDS is a detergent, it disrupts cell membrane and denatures proteins. Proteinase K helps to digest proteins, especially nucleases which can destroy DNA. Then comes phenol chloroform extraction, which separates proteins from DNA based on solubility. DNA stays in aqueous phase, proteins go into organic phase. Finally DNA is precipitated using cold ethanol.

Requirements:

• Luria Bertani (LB) Broth
• Tryptone: 10.0 g/L
• Yeast Extract: 5.0 g/L
• Sodium Chloride: 10.0 g/L
• TE Buffer (pH 8.0)
• 10 percent SDS solution
• Phenol: Chloroform: Isoamyl Alcohol (25:24:1)
• Chilled Isopropanol or 70 percent Ethanol
• 3M Sodium Acetate (pH 5.2)
• Lysozyme (10 mg/ml)
• Microcentrifuge tubes and micropipettes
• Water bath set at 37 and 60 degrees Celsius

Procedure:

1. Take 1.5 ml of overnight grown E. coli culture in a microcentrifuge tube.
2. Centrifuge at 8000 rpm for 5 minutes to pellet the cells.
3. Discard the supernatant completely and resuspend the pellet in 500 microliters of TE buffer.
4. Add 10 microliters of lysozyme and incubate at 37 degrees for 30 minutes. I have seen students skipping this, but for good yield, it is better to do it.
5. Add 50 microliters of 10 percent SDS to the tube.
6. Mix gently by inverting. Do not vortex or your DNA will break into small pieces.
7. Incubate the mixture at 60 degrees for 15 minutes until the solution becomes clear.
8. Add an equal volume of Phenol: Chloroform: Isoamyl alcohol mixture.
9. Centrifuge at 10000 rpm for 10 minutes at room temperature.
10. Carefully collect the top transparent layer into a fresh tube without touching the white protein layer in middle.
11. Add double the volume of chilled ethanol and 1/10th volume of sodium acetate.
12. Look for white thread-like precipitates appearing in the tube.
13. Centrifuge again to pellet the DNA and wash it with 70 percent ethanol.
14. Air-dry the pellet for 15 minutes and dissolve in 50 microliters of TE buffer.

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.