Plasmid Vector Construction
Plasmid Vector Construction - Related Videos
09:16
A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors
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Cited by 13 •
2016
Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.
06:25
Use of In Vivo Assembly for High-efficiency Plasmid Construction
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Cited by 2 •
2025
In vivo assembly is a ligation-independent cloning method that relies on intrinsic DNA repair enzymes in bacteria to assemble DNA fragments by homologous recombination. This protocol is both time and cost-effective, as few reagents are required, and cloning efficiency can be as high as 99 %.
12:59
High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots
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Cited by 37 •
2016
Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.
08:16
Plasmid Purification
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2023
Plasmid purification is a technique used to isolate and purify plasmid DNA from genomic DNA, proteins, ribosomes, and the bacterial cell wall. A plasmid is a small, circular, double-stranded DNA that is used as a carrier of specific DNA molecules. When introduced into a host organism via transformation, a plasmid will be replicated, creating numerous copies of the DNA fragment under study. In this video, a step-by-step generalized procedure is described for how to perform plasmid purification.
02:44
Generation of Recombinant Adeno-Associated Virus Through Plasmid Transfection
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2026
Source: Ding, J., et al. Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts. J. Vis. Exp. (2016)This video demonstrates the production of recombinant adeno-associated virus particles through plasmid transfection in mammalian cells. It highlights the coordinated expression of Rep, Cap, and helper proteins that drive viral genome replication and capsid assembly within the nucleus.
FAQs
The backbone’s origin of replication supports copying in the host cell, while its selectable marker helps distinguish cells that carry the construct from those that do not. These features affect whether recombinant DNA can be maintained and identified during experiments. Promoters and other regulatory sequences then influence how the inserted gene is used after the plasmid is propagated.
Restriction-ligation assembly and sequence-directed assembly join an insert to a plasmid backbone through different design strategies. Restriction-ligation relies on preparing compatible DNA ends before joining the pieces, whereas sequence-directed assembly uses matching sequence information to guide the connection. Choosing between them depends on how the insert and backbone are designed and on the construction workflow.
Promoters help determine whether and how strongly an inserted gene is expressed, while orientation places that gene in the intended relationship to regulatory sequences. A construct can therefore contain the correct DNA sequence yet produce an unsuitable experimental result if regulatory elements or orientation are poorly matched. Design review is essential before assembly.
Researchers select a suitable backbone, prepare the DNA insert, and join the two components by restriction-ligation or sequence-directed assembly. The resulting construct is introduced into bacterial host cells, where plasmids can be propagated and screened. This sequence links design decisions to recovery of a recombinant molecule that can support later expression or functional studies.
After introduction into bacteria, selectable markers help identify cells associated with plasmid uptake, while screening helps evaluate which recovered cells contain the intended recombinant construct. This stage is important because assembly produces candidate molecules that must be distinguished before downstream use. Reliable screening supports propagation of a plasmid suitable for expression or gene-function experiments.
It is useful when researchers need to express a protein, investigate gene function, or produce an engineered genetic material. In each case, the plasmid design connects the biological question to a controllable DNA arrangement, including the insert, promoter, and regulatory sequences. Careful construction can therefore support reproducible experiments across molecular biology and biotechnology applications.