DNA Ligation & Transformation, Mini-scale preparation of plasmid DNA Restriction enzyme & Gel electrophoresis

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1. DNA Ligation & Transformation

제한효소를 처리하여 얻어낸 insert DNA를 T4 DNA ligase를 이용하여 vector에 삽입한다. DNA strand의 5'에 존재하는 인산기와 3'에 존재하는 OH기 사이에 phosphodiester bond를 형성하여 ligation하고, ligation product를 competent cell에 형질전환한 다음 plate에 ampicillin resistance에 의해 생성된 colony를 selection한다.
2. Mini-scale preparation of plasmid DNA

Mini-prep을 통해 colony로부터 plasmid DNA를 추출하여 정제하는데, 염기성 조건에서 plasmid DNA와 chromosomal DNA의 denaturation정도의 차이와 다시 중성조건에서 두 DNA의 renaturation 정도의 차이를 이용하여 plasmid DNA를 분리해낸다.
3. Restriction enzyme & Gel electrophoresis

이후 분리한 plasmid DNA에 제한효소를 처리하여 insert DNA 와 vector를 분리하고 전기영동을 통해 확인한다. 제한효소를 이용하여 insert DNA와 vector를 분리한 다음 전기영동을 통해 확인해준 결과는 6kb와 5kb사이에서 band가 형성되었으며, 750bp에도 band가 형성되었다.

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1. DNA Ligation & Transformation

DNA ligation and transformation are fundamental techniques in molecular biology and genetic engineering. DNA ligation is the process of joining DNA fragments together using an enzyme called DNA ligase. This is an essential step in the construction of recombinant DNA molecules, which are then introduced into host cells through transformation. Transformation is the process of introducing foreign DNA into a host cell, such as bacteria or yeast, allowing the host cell to express the genes encoded in the introduced DNA. These techniques are crucial for a wide range of applications, including gene cloning, gene expression, and the production of genetically modified organisms. Mastering these techniques is essential for researchers working in fields such as biotechnology, genetics, and molecular biology.
2. Mini-scale preparation of plasmid DNA

Mini-scale preparation of plasmid DNA is a widely used technique in molecular biology and genetic engineering. Plasmids are small, circular DNA molecules that can replicate independently within a host cell, such as bacteria. Mini-scale preparation involves the isolation and purification of plasmid DNA from a small-scale bacterial culture, typically using a simple and efficient protocol. This technique allows researchers to quickly and easily obtain small amounts of high-quality plasmid DNA for various applications, such as DNA sequencing, restriction enzyme analysis, and transfection of eukaryotic cells. The ability to rapidly prepare plasmid DNA is essential for many experimental procedures and is a fundamental skill for anyone working in the field of molecular biology.
3. Restriction enzyme & Gel electrophoresis

Restriction enzymes and gel electrophoresis are two essential tools in molecular biology and genetic engineering. Restriction enzymes are enzymes that can recognize and cleave specific DNA sequences, allowing researchers to manipulate and analyze DNA molecules. Gel electrophoresis, on the other hand, is a technique used to separate and visualize DNA fragments based on their size and charge. Together, these techniques are widely used for a variety of applications, such as DNA cloning, DNA sequencing, and genetic analysis. Restriction enzymes enable the precise cutting and manipulation of DNA, while gel electrophoresis allows for the separation and visualization of the resulting DNA fragments. Mastering these techniques is crucial for researchers working in fields such as molecular biology, genetics, and biotechnology, as they provide the fundamental tools for DNA analysis and manipulation.

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