[A]서강대학교 현대생물학실험2_1차 풀레포트_PCR과 Agarose gel electrophoresis and gel purification

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1. Basic Laboratory Techniques

배지는 세포 배양을 위한 필요물질이 들어있는 것으로 LB broth와 LB agar plate를 이용한다. 수용성 세포를 형질전환 하고 선택배지로 제조된 LB agar plate를 이용해 생성된 colony로 형질전환 효율을 계산한다.
2. Competent Cell

수용성 세포(Competent cell)란, 외부의 DNA를 수용할 수 있는 상태의 박테리아로, 자연적으로 유도될 수 있지만 본 실험에서는 Ca2+ 용액 처리와 냉각과 열 충격을 주어 인위적으로 유도한다.
3. PCR (Polymerase Chain Reaction)

PCR은 총 3단계로 고온으로 DNA를 변성하고, annealing temperature에서 primer를 결합한 뒤, Taq DNA polymerase로 DNA를 합성해 Target gene을 증폭한다.
4. Agarose gel electrophoresis and gel purification

전기영동은 DNA의 크기와 전하량에 따른 분류를 가능하게 한다. PCR을 거쳐 증폭한 Mixture는 DNA조각 뿐 아니라 template DNA나 효소 등 여러가지 물질이 혼합되어 있으므로, 원하는 DNA조각만 얻는 작업이 필요하다. 이를 Gel purification이라 하며, 높은 염 농도와 낮은 pH를 갖는 buffer에 DNA를 녹이면 buffer에 존재하는 chaotropic reagent를 bridge로 DNA가 membrane에 결합하게 된다.

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1. Basic Laboratory Techniques

Basic laboratory techniques are the fundamental skills and procedures that are essential for conducting scientific research and experiments in various fields of study. These techniques include pipetting, weighing, pH measurement, solution preparation, sterilization, and proper handling of laboratory equipment and materials. Mastering these basic techniques is crucial for ensuring accurate, reliable, and reproducible results. They form the foundation upon which more complex experimental protocols and analyses are built. Proficiency in basic laboratory techniques not only enhances the quality of research but also promotes safety and efficiency in the laboratory environment. Continuous practice and adherence to standard operating procedures are necessary to maintain and improve these skills, which are vital for any scientist or researcher working in a laboratory setting.
2. Competent Cell

Competent cells, also known as transformation-competent cells, are a crucial component in molecular biology and genetic engineering. These specialized cells have the ability to take up and incorporate foreign DNA, such as plasmids or recombinant DNA constructs, through a process called transformation. Competent cells are typically prepared through various chemical or physical treatments that temporarily increase their cell membrane permeability, allowing the uptake of exogenous DNA. The ability to transform competent cells is essential for a wide range of applications, including gene cloning, protein expression, and genetic manipulation. The development and use of competent cells have significantly advanced our understanding of molecular mechanisms and enabled the creation of valuable biotechnological tools and products. Maintaining the integrity and efficiency of competent cells is crucial for successful genetic engineering experiments and the advancement of scientific research.
3. PCR (Polymerase Chain Reaction)

PCR, or Polymerase Chain Reaction, is a revolutionary molecular biology technique that has transformed the field of genetics and molecular biology. This powerful tool allows for the exponential amplification of specific DNA sequences, enabling researchers to generate large quantities of a target DNA fragment from a small initial sample. The ability to selectively amplify DNA has numerous applications, including genetic analysis, disease diagnosis, forensic identification, and evolutionary studies. PCR has become an indispensable technique in modern biology, allowing for the rapid and accurate detection, quantification, and manipulation of genetic material. The development of PCR has significantly advanced our understanding of molecular mechanisms, facilitated the advancement of various fields of study, and has become a fundamental tool in both research and clinical settings. Continuous improvements in PCR technology, such as the development of real-time PCR and digital PCR, have further expanded its capabilities and applications, making it an essential technique in the arsenal of modern molecular biology.
4. Agarose gel electrophoresis and gel purification

Agarose gel electrophoresis and gel purification are essential techniques in molecular biology and biotechnology. Agarose gel electrophoresis is a powerful method for separating and analyzing DNA, RNA, or protein molecules based on their size and charge. This technique allows researchers to visualize and quantify the size and purity of DNA fragments, which is crucial for various applications, such as gene cloning, DNA sequencing, and genetic analysis. Gel purification, on the other hand, is the process of extracting and purifying specific DNA fragments from agarose gels. This step is often necessary for downstream applications, such as ligation, transformation, or further analysis. The combination of agarose gel electrophoresis and gel purification enables researchers to isolate and purify DNA of interest, which is essential for a wide range of molecular biology experiments and techniques. These techniques have become indispensable tools in the field of molecular biology, contributing to advancements in genetic engineering, biotechnology, and our understanding of fundamental biological processes.

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