Extraction of plasmid DNA

The extraction of plasmid DNA is a basic and important experimental technique in molecular biology, which involves the process of isolating and purifying plasmid DNA from host cells such as bacteria. As the main vector for carrying foreign genes into bacteria for amplification or expression, plasmids are widely used in fields such as genetic engineering, gene therapy, and vaccine development. The following are detailed steps and precautions for plasmid DNA extraction:

1. Overview of plasmid DNA

Plasmid is an extrachromosomal stable genetic factor, usually a double-stranded, closed-circle DNA molecule, and exists in the host cell in a supercoiled state. Plasmids range in size from 1 to 200kb and are mainly found in bacteria, actinomycetes and fungal cells. They have the ability to replicate and transcribe autonomously, maintain a constant copy number in progeny cells, and express the genetic information they carry.

2. Basic steps for plasmid DNA extraction

Methods for isolating plasmid DNA from bacteria usually involve three basic steps: culturing the bacteria to amplify the plasmid, collecting and lysing the cells, and isolating and purifying the plasmid DNA. The following takes the alkaline lysis method as an example to introduce the plasmid DNA extraction process in detail:

1. Cultivation of bacteria to amplify plasmids

1>Select the appropriate bacterial strain (such as E.coli DH5α) and plasmid vector (such as pUC19).

2> Inoculate the bacteria into LB liquid medium containing appropriate antibiotics (such as ampicillin), and culture with shaking at 37°C overnight (about 12-14 hours) to amplify the plasmid in the bacteria.

2. Collect and lyse cells

1>Take a certain amount of bacterial liquid, centrifuge to remove the supernatant, and collect the bacterial sediment.

2>Use alkaline lysis to lyse bacterial cells. Commonly used reagents include solution I (containing glucose, Tris-HCl and EDTA, used to maintain the integrity of the cell membrane), solution II (containing NaOH and SDS, used to destroy the cell membrane and denature DNA) and solution III (containing KAc and glacial acetic acid , used to neutralize NaOH and precipitate proteins and chromosomal DNA).

3>Through a series of gentle operations (such as gentle turning, placing on ice, etc.), the bacterial cells are lysed and the plasmid DNA is released.

3. Isolate and purify plasmid DNA

1>Centrifuge to remove cell debris and chromosomal DNA precipitates, and collect the supernatant containing plasmid DNA.

2>Use phenol-chloroform-isoamyl alcohol mixture and other reagents to further purify the plasmid DNA to remove proteins and other impurities.

3>Recover plasmid DNA through absolute ethanol or isopropanol precipitation and dissolve it in an appropriate buffer (such as TE buffer).

3. Precautions

1. Operating specifications: During the entire extraction process, aseptic operating specifications should be strictly followed to avoid contamination of exogenous DNA.

2. Reagent selection: Choose high-quality reagents and consumables to ensure the accuracy and repeatability of the experiment.

3. Condition control: Pay attention to controlling the experimental conditions (such as temperature, time, pH value, etc.) to obtain the best extraction effect.

4. Safety protection: When using toxic or harmful reagents, personal protection and laboratory safety measures should be taken.

4. Summary

The extraction of plasmid DNA is an important experimental technique in molecular biology. The process involves multiple steps such as bacterial culture, cell lysis and DNA purification. Through strict operating specifications and condition control, high-purity, high-quality plasmid DNA can be obtained, providing strong support for subsequent experimental research.