Publish Time: 2024-06-11 Origin: Site
Plasmid extraction is one of the commonly used techniques in molecular biology experiments. Although the operation is simple, many people do not fully understand the principles behind it. Commonly used plasmid extraction kits on the market usually extract pure plasmid DNA by adding different solutions (such as solutions 1, 2, and 3), followed by washing and elution steps. However, the components of each solution and the specific role they play in the experiment are still unfamiliar to many people.
Plasmid extraction experimental principle
Principle
In cells, there are a variety of biological macromolecules, including proteins, genomic DNA, plasmid DNA, and RNA. The goal of plasmid extraction is to isolate pure plasmid DNA from these complex molecular mixtures without interference from other macromolecules. To achieve this, specific methods are needed to treat these different molecules individually.
Genomic DNA and plasmids
Distinguish
Bacteria do not have a nucleus, only a nucleoid. The DNA is circular, but not naked, and has a variety of nucleoproteins (NAPs) bound to it. NAPs and gene transcription activity can change the morphological structure of the nucleoid. Relatively speaking, plasmid DNA is relatively simple and is free in the cytoplasm in the form of covalently closed circular (cccDNA).
Common plasmid extraction methods
Method
Currently commonly used plasmid extraction methods include alkaline lysis, boiling and detergent lysis. The first two methods are more vigorous and suitable for smaller plasmids (<15Kb), while the detergent lysis method is gentler and suitable for larger molecular weight plasmids (>15Kb).
Alkaline lysis method
Alkaline lysis is a relatively common plasmid DNA extraction method, which is based on the topological differences between covalently closed circular DNA and linear DNA. The steps are as follows:
1) Alkaline lysis and cell rupture
In a strongly alkaline environment (pH 12.0-12.6), bacterial cell walls and cell membranes are destroyed by sodium dodecyl sulfate (SDS), and genomic DNA and plasmid DNA are released.
Chromosomes and linear DNA double helix structures are denatured under highly alkaline conditions, while plasmid DNA is not easily denatured due to its compact structure.
2) Neutralization and restoration
By adjusting the pH of the solution to neutral, the covalently closed circular plasmid DNA can quickly renature and restore its natural structure.
Linear genomic DNA renatures slowly, becomes entangled with proteins and cell debris, forms insoluble complexes, and precipitates.
3) Precipitation and separation
Under high-salt conditions (e.g., using potassium ions instead of sodium ions), cell debris, denatured proteins, and linear genomic DNA precipitate, while plasmid DNA remains soluble.
After centrifugation, the precipitate is removed, and the plasmid DNA in the supernatant can be further purified by ethanol precipitation or specific adsorption on silica gel membrane.
Main reagent ingredients and functions
Composition and Function
Before there was a plasmid extraction kit, most of the plasmid extraction kits used reagents to extract plasmids from E. coli. The following three reagents were mainly used:
Lysate
Glucose: Increases solution viscosity, maintains osmotic pressure, and prevents DNA from being degraded due to mechanical shearing force.
EDTA: chelates metal ions such as Mg⊃2;⁺ and Ca⊃2;⁺, inhibits the degradation of DNA by deoxyribonuclease (DNase), and contributes to the action of lysozyme.
NaOH-SDS liquid
NaOH: Nucleic acid is stable in solutions with pH > 5 and < 9, but at pH > 12 or < 3, it will cause dissociation of hydrogen bonds between double strands and cause denaturation.
SDS: SDS is an ionic surfactant. Its main functions are: 1. Dissolve lipids and proteins on cell membranes, thus dissolving membrane proteins and destroying cell membranes; 2. Depolymerizing nuclear proteins in cells; 3. SDS can combine with proteins to form RO-SO3-...R+-protein complexes, causing the proteins to denature and precipitate.
3mol/L NaAc (pH4.8) solution
This solution is a NaAc-HAc buffer, which adjusts the pH of the extraction solution to neutral so that the denatured plasmid DNA can renature and exist stably.
High-salt 3 mol/L NaAc is conducive to the aggregation and precipitation of denatured macromolecule chromosomal DNA, RNA and SDS-protein complexes.