Experiment: Extraction of Genomic DNA from E. coli

Purpose

The purpose of this experiment is to isolate high-quality genomic DNA from Escherichia coli (E. coli) cells. This DNA will be free of contaminants such as proteins, RNA, and salts. Purified genomic DNA can be used in various downstream applications, including PCR, sequencing, and genetic analysis. This experiment provides hands-on experience with DNA extraction techniques and introduces students to important principles in molecular biology.

Principle

DNA extraction involves breaking down cell membranes to release DNA into solution, removing cellular proteins, and purifying DNA from contaminants. This protocol uses a column-based method that leverages the binding properties of DNA under high-salt conditions. Key reagents include:

1. Lysis Buffers (Buffer GA and Buffer GB): These buffers help in breaking down the cell membrane and inactivating nucleases that can degrade DNA. Buffer GA also creates an ideal pH and salt concentration for enzymatic digestion.

2. Proteinase K: This enzyme digests proteins, helping to release DNA from the cell structure while eliminating proteins that could contaminate the DNA sample.

3. Ethanol: Facilitates DNA precipitation and binding to the column matrix.

4. Wash Buffers (Buffer PB and Buffer PW): Remove remaining proteins, RNA, and salts from the DNA, ensuring a clean sample.

5. Elution Buffer: Releases the DNA from the column into a purified solution.

Through centrifugation, DNA is bound to a silica-based column membrane under high-salt conditions and later eluted as a purified product.

Materials

• Equipments:

  • E. coli cell culture (1 ml), 1.5 ml microcentrifuge tubes, Pipettes and pipette tips, Centrifuge (capable of 12,000 rpm), Water bath set at 70°C, FastPure gDNA Mini Columns I, Collection tubes (2 ml), Electrophoresis apparatus (optional for verifying DNA quality), Agarose gel (optional for DNA visualization), DNA ladder (optional for size comparison)

• Reagents:

  • Buffer GA: Provides an environment suitable for enzymatic hydrolysis of cells.

  • Proteinase K: Enzymatically digests proteins and aids in cell lysis.

  • Buffer GB: Inactivates Proteinase K and provides optimal conditions for DNA binding.

  • Buffer PB: Removes residual proteins and RNA from the DNA sample.

  • Buffer PW: Removes salts and other small contaminants from the DNA.

  • Anhydrous ethanol: Facilitates DNA binding to the column.

  • Elution Buffer: Releases purified DNA from the column.

Procedures

A. Sample Processing

1. Preparation of Cell Sample a. Take 1 ml of E. coli culture in a 1.5 ml centrifuge tube. b. Centrifuge at 10,000 rpm for 1 minute. Discard the supernatant, leaving a cell pellet.

2. Lysis and Digestion a. Add 230 µl of Buffer GA to the cell pellet. Vortex until the cells are fully resuspended. b. Add 20 µl of Proteinase K to the solution and mix thoroughly. c. Add 250 µl of Buffer GB, then vortex and incubate the mixture at 70°C for 10 minutes.

   Note: If a white precipitate forms, ensure it dissolves by vortexing again or continuing incubation. Complete lysis is essential for maximum DNA yield. d. Add 180 µl of anhydrous ethanol, vortex to mix. A cloudy or flocculent precipitate may appear, which is normal.

B. DNA Binding and Purification

1. Binding to Column a. Transfer the entire mixture to the FastPure gDNA Mini Column I (placed in a collection tube). b. Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through.

2. Wash with Buffer PB a. Add 500 µl of Buffer PB to the column and centrifuge at 12,000 rpm for 1 minute. Discard the filtrate.

3. Wash with Buffer PW a. Add 600 µl of Buffer PW to the column and centrifuge at 12,000 rpm for 1 minute. Discard the filtrate. b. Centrifuge the empty column at 12,000 rpm for an additional 2 minutes to remove any residual ethanol. c. Leave the column open for 2 minutes to air-dry if needed. This step ensures complete removal of ethanol, which can inhibit downstream applications.

C. Elution of DNA

1. Eluting the DNA a. Place the column in a new, labeled 1.5 ml microcentrifuge tube. b. Add 50 µl of Elution Buffer to the center of the column membrane. c. Incubate at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 1 minute. d. The flow-through contains the purified genomic DNA.

Results

After completing the extraction, you should have a clear, purified DNA solution in the collection tube. To verify the DNA quality and quantity:

• UV Spectrophotometry: Measure absorbance at 260 nm and 280 nm to check DNA concentration and purity. A 260/280 ratio of ~1.8 indicates pure DNA.

• Agarose Gel Electrophoresis (Optional): Load 5 µl of the eluted DNA with a DNA ladder on an agarose gel. Electrophoresis will allow visualization of the DNA band and comparison with known sizes in the DNA ladder.

Discussion

Why Each Step is Important?

• Lysis and Proteinase K: Efficient cell lysis and protein digestion are crucial for releasing DNA without contamination. Incomplete lysis or insufficient Proteinase K activity would reduce DNA yield and purity.

• Buffer GB and 70°C Incubation: Heating helps dissolve cell debris and enhance lysis. It’s essential to ensure all materials dissolve during this step to avoid impurities.

• Ethanol Addition: Ethanol causes DNA to precipitate and bind to the column, enabling its separation from other cell components.

• Washing Steps (Buffers PB and PW): These washes remove proteins, RNA, and salts, which could otherwise inhibit DNA-based applications.

• Elution Buffer: This buffer efficiently releases DNA from the silica column, providing a pure DNA solution.