Isolation of Plasmid DNA Using Alkaline Lysis Method
The alkaline lysis method is the most common technique for isolating plasmid DNA. The technique helps separate plasmid DNA from chromosomal DNA.
The method for plasmid DNA separation is widely used for DNA extraction. Along with DNA extraction, the technique enables the purification of plasmid DNA from bacterial cells.
The alkaline lysis method is useful in molecular biology. The method has various applications including cloning, sequencing, and transformation.
This method of Plasmid DNA separation is efficient and relatively quick. This blog will help you to gain a deeper understanding of the Alkaline Lysis Method.
Isolation Of Plasmid DNA By Alkaline Lysis Method Principle
The alkaline lysis method is a common technique for isolation of plasmid DNA. It allows the efficient extraction and purification of plasmid DNA from bacterial cells.
The method is a powerful and highly effective tool and yields high-quality DNA with precision. The extracted plasmid DNA finds its use in various molecular biology applications, like cloning, sequencing, and transformation.
The principle of the alkaline lysis method for separating plasmid DNA involves selectively lysing bacterial cells and denaturing chromosomal DNA. The technique keeps the plasmid DNA intact throughout the process.
The principle of plasmid DNA separation from the alkaline lysis method is given below:
- Cell Lysis – Cell lysis involves buffering and lysis solution. In cell lysis, the cell membrane disruption allows the release of internal contents. The contents include organelles, proteins, and nucleic acids.
Cell lysis helps study cellular components, DNA, RNA, and proteins. The method is performed through mechanical methods, chemical methods, or enzymatic methods. Selecting a suitable method depends on the type of cell and the specific components to be extracted. Lysis solutions like sodium dodecyl sulfate (SDS) and sodium hydroxide (NaOH) for denaturation. The denaturation of plasmid DNA and chromosomal DNA is done using the solutions.
- Denaturation – The process of denaturation of the chromosomal DNA and plasmid DNA happens with NaOH. The solution has a high pH, that facilitates the denaturation process.
NaOH leads to the entanglement of precipitation of the single-stranded DNA molecules. The denaturation of protein leads to the formation of insoluble complexes.
- Neutralization – The process involves the use of solutions like potassium acetate (KOAc) or acetic acid. The addition of solution neutralizes the pH, thus bringing it back to neutral.
During the process, the plasmid DNA stays in the solution, as they are of smaller size. Denatured proteins and larger chromosomal DNA aggregate and form precipitation.
- Centrifugation – The mixture of plasmid DNA and chromosomal DNA is passed to the centrifugation process.
The process separates the precipitated chromosomal DNA, proteins, and cell debris (pellet) from the plasmid DNA (supernatant).
- Plasmid DNA Purification – Plasmid DNA undergoes purification through processes like ethanol precipitation, phenol-chloroform extraction, or column-based purification.
Firstly, plasmid DNA is collected for purification. Such processes help remove any remaining impurities in the plasmid DNA.
Isolation Of Plasmid DNA By Alkaline Lysis Method Experiment
Separation of plasmid DNA from chromosomal DNA through the alkaline lysis method is a common laboratory technique. The method helps extract plasmid DNA from bacterial cells.
The method exploits the differential denaturation and renaturation properties of plasmid and chromosomal DNA under alkaline conditions.
The method of extraction uses the alkaline lysis method. Here is a detailed experimental procedure for separating plasmid DNA and chromosomal DNA:
Material Required
- Bacterial culture (E. coli with plasmid)
- Alkaline lysis buffers (Solution I, Solution II, Solution III)
- Centrifuge and microcentrifuge tubes
- RNase A
- Phenol/chloroform/isoamyl alcohol (optional)
- Ethanol or isopropanol
- TE buffer (Tris-EDTA)
Isolation Procedure
- Culture Preparation and Cell Harvesting – The bacterial culture is grown overnight in an LB medium. The appropriate antibiotic is added to ensure plasmid maintenance.
The process involves harvesting 1.5 mL of the culture through centrifugation at 12,000 x g for 1 minute. The next step is to discard the supernatant.
- Resuspension –The bacterial pellet is suspended in 100 µL of Solution I. This involves pipetting up and down or vortexing until the pellet is completely dissolved.
- Lysis – 200 µL of Solution II must be added to the re-suspended cells. The mixture must be mixed gently by inverting the tube 4-6 times. Do not vortex the mixture.
The mixture must be incubated at room temperature for 5 minutes. The step lyses the cells and denaturizes both plasmid and chromosomal DNA.
- Neutralization –150 µL of Solution III must be added to the mixture. The solution needs to be gently mixed by inverting the tube until the solution becomes milky.
The mixing neutralizes the solution, causing plasmid DNA to remain in the solution. The chromosomal DNA and proteins form a precipitate. The tube on ice needs to be incubated for 5-10 minutes.
- Centrifugation – The next step is to centrifuge the tube at 12,000 x g for 10 minutes at 4°C. The process will lead to the formation of a pellet of chromosomal DNA and cellular debris. After centrifugation, plasmid DNA will remain in the supernatant.
- Supernatant Collection – the clear supernatant solution must be transferred to a new microcentrifuge tube. Make sure you do not disturb the pellet.
- Plasmid DNA Precipitation – A small amount of isopropanol is added to the supernatant. Gently mix the solution by inverting the tube. To precipitate the plasmid DNA, incubate the mixture at room temperature for 10 minutes.
- Wash – The pellet must be washed with ethanol and centrifuge at 12,000 x g for 5 minutes.
- Drying – The pellet is then allowed to air dry or use a vacuum desiccator. This removes any remaining ethanol.
- Resuspension
- Storage – The plasmid DNA is stored at -20 °C until used.
The Link Between Isolation Of Plasmid DNA And Genetic Testing
Isolation of plasmid DNA is a crucial step in many genetic testing and molecular biology applications. Small circular DNA molecules like Plasmid DNA are present in bacteria.
Plasmid DNA replicates independently of chromosomal DNA. Plasmid DNA has a significant role in molecular biology and genetic testing. This is so, because of their ability to carry foreign genes and replicate them within host cells.
Conclusion
The alkaline lysis method for plasmid DNA isolation from bacterial cells is a reliable and efficient technique. The steps involved in DNA separation include cell lysis, neutralization, and DNA precipitation.
The steps lead to selective extraction of plasmid DNA. They help remove chromosomal DNA and proteins. The purified plasmid DNA can then be used for various applications, including cloning, sequencing, and genetic testing.