PlasmidDNA Purification & Sanger Sequencing 보고서

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1. Bacterial transformation

박테리아 transformation은 특정 DNA 조각을 박테리아의 유전체 내로 삽입하여 새로운 유전형질이 발현되도록 하는 방법입니다. 박테리아 plasmid는 restriction enzyme이 작용하는 multicloning site와 항생제 내성 유전자를 가지고 있어 외부 유전 물질을 자유롭게 삽입할 수 있습니다. 이를 통해 약 8kb의 긴 DNA 조각을 박테리아 plasmid에 삽입하여 복제할 수 있습니다.
2. Liquid LB culture 및 Colony inoculation

원하는 균주를 Luria Broth(LB) 배지에서 배양하여 충분히 성장시킨 후, 생성된 single colony를 분리하여 다시 liquid LB에 옮겨 배양하는 과정입니다. 이를 통해 많은 양의 plasmid DNA를 얻을 수 있습니다. LB 배지는 박테리아 세포의 단백질 합성, 비타민, 미네랄 등을 제공하여 세포 성장을 돕습니다.
3. Plasmid purification

박테리아 배양에서 plasmid DNA를 분리, 정제하는 방법입니다. 주로 Plasmid MiniPrep Kit를 사용한 Alkaline lysis 방법을 통해 plasmid DNA를 추출합니다. 이 과정에서 사용되는 버퍼들의 역할은 다음과 같습니다: PA1 버퍼는 세포벽 파괴와 RNA 분해, PA2 버퍼는 단백질 변성과 DNA 변성, PA3 버퍼는 pH 중화와 plasmid DNA 재결합, PB 버퍼는 DNA와 resin 사이의 결합, W2 버퍼는 DNA 순도 증가를 위한 세척 등입니다.
4. Sanger Sequencing

Sanger sequencing은 ddNTP 염기를 이용한 DNA 염기서열 분석 방법입니다. ddNTP는 DNA 중합 반응을 중단시켜 각 염기의 위치를 확인할 수 있습니다. 이 방법은 개별 유전자 또는 작은 DNA 조각의 서열을 확인하는 데 유용하지만, 최근에는 차세대 염기서열 분석법(NGS)이 더 많이 사용되고 있습니다.
5. Sequence 확인 및 BLAST

Sanger sequencing 결과를 Chromas 프로그램으로 확인하여 raw quality를 점검할 수 있습니다. 또한 NCBI BLAST 프로그램을 통해 sequencing 결과와 reference sequence 간의 일치율을 확인할 수 있습니다. 이를 통해 DNA 증폭과 분리가 제대로 진행되었는지 확인할 수 있습니다.

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1. Bacterial transformation

Bacterial transformation is a fundamental technique in molecular biology and biotechnology. It involves the introduction of foreign DNA into bacterial cells, allowing them to express new genetic traits. This process is crucial for various applications, such as gene cloning, protein expression, and genetic engineering. The ability to transform bacteria opens up a world of possibilities, from the production of therapeutic proteins to the development of genetically modified organisms for research and industrial purposes. Understanding the mechanisms and optimizing the conditions for successful bacterial transformation is an essential skill for any scientist working in these fields. By mastering this technique, researchers can unlock new avenues of discovery and innovation, pushing the boundaries of what is possible in the life sciences.
2. Liquid LB culture 및 Colony inoculation

Liquid LB (Luria-Bertani) culture and colony inoculation are fundamental techniques in microbiology and molecular biology. Liquid LB culture allows for the growth and propagation of bacterial cells, providing a controlled environment for their cultivation. This is a crucial step in many experimental procedures, as it enables the generation of large quantities of bacterial cells for various downstream applications, such as plasmid purification, protein expression, and genetic manipulation. Colony inoculation, on the other hand, involves the transfer of individual bacterial colonies from solid growth media to liquid culture, ensuring the isolation and amplification of specific strains or genetic constructs. These techniques are essential for maintaining and expanding bacterial cultures, as well as for ensuring the purity and consistency of experimental samples. Mastering these skills is crucial for any researcher working with bacteria, as they form the foundation for a wide range of molecular biology and biotechnology experiments.
3. Plasmid purification

Plasmid purification is a crucial technique in molecular biology and genetic engineering. Plasmids are circular DNA molecules that can be isolated from bacterial cells and used for a variety of applications, such as gene cloning, protein expression, and genetic manipulation. The ability to efficiently purify plasmids is essential for many experimental procedures, as it allows researchers to obtain high-quality, pure DNA samples that can be used for downstream applications. Plasmid purification involves a series of steps, including cell lysis, DNA extraction, and purification, to isolate the plasmid of interest from the bacterial host. Mastering this technique is crucial for researchers working in fields such as molecular biology, biotechnology, and synthetic biology, as it enables the generation of the necessary genetic tools and constructs for their experiments. By optimizing plasmid purification protocols, researchers can ensure the reliability and reproducibility of their results, ultimately advancing the field of genetic engineering and its applications.
4. Sanger Sequencing

Sanger sequencing is a fundamental technique in molecular biology and genetics, providing a reliable method for determining the precise order of nucleotides in a DNA sequence. This method, developed by Frederick Sanger in the 1970s, has been a cornerstone of genetic research, enabling the identification and characterization of genes, the analysis of genetic variations, and the study of genomic structures. Sanger sequencing involves the controlled synthesis of DNA fragments using chain-terminating nucleotides, which are then separated and detected to reveal the sequence of the target DNA. Despite the emergence of newer sequencing technologies, such as next-generation sequencing, Sanger sequencing remains an essential tool for various applications, including DNA cloning, mutation analysis, and the validation of sequencing results. Mastering Sanger sequencing techniques is crucial for researchers working in fields like molecular biology, genetics, and genomics, as it allows them to generate high-quality, reliable sequence data that can be used to drive scientific discoveries and advancements.
5. Sequence 확인 및 BLAST

Sequence confirmation and BLAST (Basic Local Alignment Search Tool) analysis are essential steps in the process of genetic research and molecular biology. Sequence confirmation involves verifying the accuracy of a DNA sequence, ensuring that the genetic information obtained matches the expected or desired sequence. This step is crucial for validating the integrity of experimental results, as well as for identifying any potential errors or mutations that may have occurred during the sequencing process. BLAST, on the other hand, is a powerful bioinformatics tool that allows researchers to compare a given DNA or protein sequence against a comprehensive database of known sequences. By performing BLAST analysis, researchers can identify similar sequences, determine evolutionary relationships, and gain insights into the potential functions and characteristics of the target sequence. Mastering these techniques is vital for researchers working in fields such as genomics, molecular biology, and bioinformatics, as they enable the accurate identification, characterization, and interpretation of genetic information. The ability to confirm sequences and leverage BLAST analysis is a crucial skill that underpins many scientific discoveries and advancements in the life sciences.