Biological Molecule Experiment Textbook

Author: Zhai Zhaoqiang
Compiler/Origin:
Publisher:
Publish Date: 2004-06-01
Features: Plasmid DNA Extraction
[Principle] Plasmid DNA is an independent molecule within the cell, consisting of double-stranded circular DNA without proteins. It primarily exists in prokaryotic cells and plant cells. Plasmid DNA is free in the cytoplasm, separate from chromosomal DNA, and does not integrate with it. It exists in multiple copies. Since plasmid DNA can replicate independently within the cell without relying on cell division or chromosomal control, it is widely used in molecular biology as a vector for carrying exogenous DNA. This involves using molecular cloning techniques to insert exogenous DNA fragments or genes into the plasmid DNA molecule, followed by transformation into host cells to obtain clones carrying the exogenous gene or DNA fragment (clonal line). Through the rapid proliferation of host cells and the replication of the plasmid itself within the cell, large copies of the exogenous gene or DNA fragment in the clones can be obtained, and the exogenous gene can be expressed as a protein in the host cells. Therefore, based on the two purposes of using plasmids as DNA vectors, they can be divided into two categories: one is plasmid DNA molecules used solely for cloning and amplifying exogenous DNA fragments or genes, known as cloning vectors (cloning plasmids); the other is plasmids equipped with protein expression regulatory elements (usually promoter structures) at appropriate positions upstream of the multiple cloning site on the plasmid DNA molecule, known as expression vectors (expression plasmids). Based on the nature of the expression regulatory elements (eukaryotic or prokaryotic), expression plasmids are further divided into eukaryotic expression plasmids or prokaryotic expression plasmids. As a vector molecule, the plasmid used in cloning should meet several essential conditions: (1) It must have an origin of replication capable of replicating within the cell to ensure autonomous replication in the host cell; (2) It must have multiple single restriction enzyme recognition sites that can ligate with exogenous DNA, allowing exogenous DNA and the vector molecule to be fully cut and ligated, forming a recombinant plasmid DNA molecule; (3) The vector molecule should have selectable markers, such as antibiotic resistance genes, enzyme genes, or nutritional auxotrophic genes; (4) The molecular size of the plasmid vector should not be too large to accommodate a certain size of exogenous DNA fragments. Cloning vectors are required to produce large copies of exogenous DNA through replication, so such vectors should have higher copy numbers in appropriate host cells. For expression vectors, they need to be equipped with promoters, leader sequences, polyadenylation signals, and enhancers upstream of the multiple cloning insertion site that are compatible with the host cells. Some expression vectors also include signal peptide genes or fusion protein gene fragments to facilitate expression detection or purification of the expression product. Plasmid DNA extraction is a fundamental operation technique in molecular biology and a cornerstone of molecular biology techniques. The preparation of plasmid DNA molecules serves different purposes. To obtain pure plasmid DNA for molecular cloning, protein expression product extraction and purification, or for transfection of host cells, a large culture of bacteria carrying the plasmid is required to extract a sufficient amount of plasmid DNA for use. Small-scale plasmid DNA extraction is often used for cloning identification, where several colonies or cell clones are picked from the transformed plates or transfected cell culture plates for verification. The colonies or cell clones are extracted using plasmid DNA extraction methods and directly subjected to electrophoresis or enzymatic digestion to determine whether they carry the plasmid and whether there is exogenous DNA inserted into it. If present, it indicates successful recombination; if not, more plasmid DNA needs to be extracted from additional clones for verification. Small-scale plasmid DNA extraction is advantageous for its speed and simplicity. One person can often simultaneously verify multiple clones at once. Small-scale plasmid DNA extraction is a fundamental step for beginners in molecular biology experiments. A significant amount of molecular biology research revolves around nucleic acid molecules. Mastering this technique is essential for anyone working in molecular biology.
[Methods]
1. Cultivation of host cells carrying plasmids: Using an inoculating loop, sterilize it by heating in a flame and then cool it. Pick a single colony from the bacterial plate and inoculate it into a medium test tube containing 2–3 mL of LB medium. Shake the culture at 37°C for 12 hours on a shaker; or directly dip a small amount of bacteria from the preserved liquid culture into the medium test tube and shake at 37°C for 12 hours; or use an inoculating loop to scrape a single colony from the cultured bacterial plate and inoculate it into an Eppendorf tube containing 1.5 mL of TE buffer.
2. Take 1.5 mL from the overnight culture and transfer it into an Eppendorf tube. Centrifuge at 12,000 rpm for 30 seconds using a tabletop centrifuge. If the bacteria were scraped, centrifuge directly.
3. Discard the supernatant and place the tube upside down on a piece of laboratory paper to allow the culture liquid to drain.
4. Add 100 μL of Solution I and suspend the pellet thoroughly by vortexing or by repeatedly aspirating and expelling with a pipette to ensure complete suspension. Let it stand at room temperature for 5 minutes.
5. Add 200 μL of Solution II (prepared freshly) and gently invert the tube 3–5 times to mix the liquid thoroughly. Incubate on ice for 5 minutes.
6. Add 150 μL of Solution III and vortex the tube vigorously twice to mix the liquid. Incubate on ice for 3 minutes.
7. Centrifuge at 12,000 rpm for 5 minutes (at room temperature or 4°C).
8. Transfer the supernatant to another Eppendorf tube using a pipette. Add twice the volume of absolute ethanol and invert the tube to mix thoroughly. Centrifuge at 12,000 rpm for 10 minutes (at room temperature).
9. Discard the supernatant and place the tube upside down on laboratory paper to allow it to dry as much as possible (about 5 minutes).
10. Add 15 μL of TE buffer and dissolve the pellet at the bottom for electrophoresis detection.
Enzyme-linked immunosorbent assay (ELISA) is an immunodetection technique developed on the basis of immunoenzymatic techniques. It has now become a routine diagnostic tool in clinical medicine for detecting antibodies, antigens, or haptenes. The ELISA technique involves immobilizing antibodies (antigens) on a solid-phase carrier (also called coating), adding the test antigen (antibody), and then reacting it with a corresponding enzyme-labeled antibody (antigen) to form specific antigen-antibody complexes. Finally, the enzyme substrate is added to produce a colored product. Since the amount of the test antigen (antibody) is proportional to the colored product formed, the antigen (antibody) concentration can be calculated based on the absorbance. The enzyme conjugate is the product of the linkage between enzymes and antibodies or antigens/haptens under the action of a cross-linking agent. It is a critical reagent for the success of ELISA. ELISA not only exhibits specific immunological reactions between antigens and antibodies but also shows enzymatic reactions, thereby demonstrating biological amplification. The prepared enzyme conjugate must meet three conditions: high purity, high activity, and monovalency. The enzyme should have stable performance, be economical and readily available, not produce a color reaction with the substrate when used alone, only produce a colored product at the end point, and be easily detectable. Commonly used enzymes include alkaline phosphatase, horseradish peroxidase (HRP), glucose oxidase, and β-galactosidase (Table 7–1). They can catalyze the conversion of colorless substrates into colored products and have specific absorption peaks. After the enzymatic reaction is terminated, the substrate remains unchanged.