Plasmid extraction is one of the cornerstone technologies in the field of molecular biology. Its core is to accurately isolate plasmid DNA from bacterial cells (especially common hosts such as Escherichia coli). Plasmid, this unique circular small molecule DNA structure, is not only an indispensable vector tool in genetic engineering, but also plays an important role in genetic information transfer, gene expression regulation and biotechnology applications because of its stability and operability. status.
1. Principle of plasmid extraction The principle of plasmid extraction is mainly based on the difference in denaturation and renaturation of DNA as well as the difference in physical and chemical properties. The specific principles are as follows: 1. Denaturation and renaturation of DNA: (1) Under alkaline conditions, both plasmid DNA and chromosomal DNA will be denatured, that is, the double strands will unwind into single strands. However, because plasmid DNA is a covalently closed circular structure, its degree of denaturation is relatively low. (2) When the pH value is adjusted to neutral, plasmid DNA can quickly renature and re-form a double-stranded structure. However, chromosomal DNA cannot completely renature due to its large molecular weight and complex structure, forming an insoluble network structure and precipitating. . 2. Differences in physical and chemical properties: (1) There are differences in molecular weight, conformation, charge, etc. between plasmid DNA and chromosomal DNA. These differences cause them to exhibit different physical and chemical properties under different conditions. (2) By adjusting the pH value, salt concentration, temperature and other conditions of the solution, effective separation of plasmid DNA and chromosomal DNA can be achieved. 3. Purification effect of organic solvents: (1) Organic solvents such as phenol/chloroform can remove residual proteins and other impurities and further purify plasmid DNA. These organic solvents can destroy the hydrophobic bonds and hydrogen bonds of proteins, causing them to denature and precipitate without damaging the structure of plasmid DNA.
2. Plasmid extraction process using Opentrons instruments As a company specializing in automated laboratory instruments, Opentrons provides instruments and solutions that are widely used in the field of molecular biology, including plasmid extraction. However, it should be noted that Opentrons does not directly produce a single instrument specifically for plasmid extraction, but rather through its automated pipetting workstations (such as Opentrons OT-2) and corresponding functional modules (such as magnetic bead purification modules). Supports the automation of experimental operations such as plasmid extraction. When using Opentrons instruments for plasmid extraction, you can follow the following general steps (specific steps may be adjusted depending on experimental requirements and kits used): 1. Experimental preparation: (1) Place the Opentrons OT-2 pipetting workstation And its corresponding functional modules (such as magnetic bead purification module) are installed in the laboratory, and the necessary calibration and settings are performed. (2) Prepare experimental consumables such as kits, test tubes, centrifuge tubes, etc. required for plasmid extraction, and ensure that they are compatible with Opentrons instruments. 2. Sample processing: (1) According to the instructions of the plasmid extraction kit, culture, collect, lyse, etc. the bacteria containing plasmids. These steps typically need to be performed manually on a traditional bench, but can also be automated using Opentrons' instrumentation when possible. 3. Automated extraction: (1) Transfer the processed sample to the Opentrons OT-2 pipetting workstation, and install the magnetic bead purification module (if the magnetic bead method is used for plasmid purification). (2) Write or select an appropriate automated program that will guide Opentrons OT-2 to perform operations such as pipetting, mixing, and magnetic separation to complete the plasmid purification process. (3) The automated program will utilize the precise pipetting capabilities of Opentrons OT-2 and the efficient purification capabilities of the magnetic bead purification module to isolate plasmid DNA from the lysate. 4. Subsequent processing: (1) After the automated procedure is completed, transfer the purified plasmid DNA to an appropriate container and perform necessary subsequent processing (such as washing, drying, etc.). (2) Use appropriate detection methods (such as electrophoresis, spectrophotometer, etc.) to conduct quantitative and qualitative analysis of the extracted plasmid DNA.
Plasmid extraction, a basic technology in molecular biology, is not only a bridge between laboratory research and genetic engineering applications, but also a key force in promoting the continuous advancement of the field of life sciences. The principle of plasmid extraction is based on the differences in physical and chemical properties of DNA molecules under specific conditions, and cleverly achieves the goal of isolating and purifying target DNA molecules from complex biological systems. The carefully designed extraction steps ensure the accuracy, efficiency and repeatability of the entire process, laying a solid foundation for subsequent genetic manipulation and analysis.
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