[서강대학교 현대생물학실험2] (2차 풀레포트) TA cloning & transformation, mini-prep, Enzyme cut, electrophoresis

본 내용은 " [서강대학교 현대생물학실험2] (2차 풀레포트) TA cloning & transformation, mini-prep, Enzyme cut, electrophoresis " 의 원문 자료에서 일부 인용된 것입니다. 2024.09.11

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• 1. TA cloning

  TA cloning을 통해 insert를 삽입한 vector는 transformation을 이용해 세포에 주입하여 lacZ selection으로 insert 삽입 여부를 확인할 수 있다. lacZ operon이 작동하면 β-1, 4-linkage를 분해 시킬 수 있는 β-galactosidase가 생성된다. X-gal(5-bromo-4-chloro-3-indolyl-D-galactopyranoside)은 β-1, 4 linkage를 가지고 있으며 이 linkage가 분해되면 파란색을 띠게 된다.

• 2. Transformation

  본 실험은 LB agar plate에 Xgal과 IPTG(Isopropyl-β-D-thiogalactopyranoside)를 spreading하여 vector를 주입한 대장균을 배양하였다. 밤새 배양한 결과, plate에서 하얀색 colony와 파란색 colony를 확인할 수 있었다.

• 3. Mini-prep

  Vector를 삽입한 대장균에는 plasmid DNA(deoxyribo nucleic acid)뿐만 아니라 chromosomal DNA를 함께 가지고 있어 mini-prep(mini-scale preparation of plasmid DNA)를 통해 plasmid를 따로 분리할 수 있다. Alkaline 조건에서 chromosomal DNA은 완전히 denaturation되지만, plasmid DNA는 완전히 denaturation되지 않고 중성에서 다시 renaturation된다.

• 4. Enzyme cut

  전기 영동 결과 insert를 삽입한 vector가 insert로 인해 크기가 커져 insert가 삽입되지 않은 sample보다 높은 위치에서 band가 형성되었다.

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• 1. TA cloning

  TA cloning is a widely used molecular biology technique that allows for the efficient insertion of DNA fragments into plasmid vectors without the need for restriction enzyme digestion and ligation. This method takes advantage of the terminal transferase activity of certain DNA polymerases, which add a single deoxyadenosine (A) residue to the 3' ends of PCR products. Complementary single-stranded 'T' overhangs on the plasmid vector then allow for the annealing and ligation of the PCR product, creating a recombinant plasmid. TA cloning is a simple, fast, and cost-effective approach that is particularly useful for cloning PCR amplicons, as it eliminates the need for restriction site engineering and the associated optimization steps. However, it is important to note that TA cloning can be less specific than traditional restriction-based cloning, as the A-T base pairing is not as stringent as the complementary base pairing required in restriction enzyme-based methods. Overall, TA cloning remains a valuable tool in the molecular biologist's toolkit, providing a convenient and efficient way to clone DNA fragments into plasmid vectors.

• 2. Transformation

  Transformation is a fundamental technique in molecular biology that involves the introduction of foreign genetic material, such as plasmid DNA, into a host cell. This process allows researchers to manipulate and study the behavior of genes and proteins within a controlled cellular environment. The most common method of transformation involves the use of chemically competent bacterial cells, typically Escherichia coli, which are treated with calcium chloride or other agents to temporarily increase their cell membrane permeability. The plasmid DNA is then added to the competent cells, and the mixture is subjected to a brief heat shock, which facilitates the uptake of the DNA. Successful transformation events can then be selected for using antibiotic resistance markers encoded on the plasmid. Transformation is a crucial step in many molecular biology workflows, enabling the amplification, modification, and expression of recombinant DNA constructs. It is a versatile technique that has numerous applications, from basic research to biotechnology and genetic engineering. As such, a thorough understanding and mastery of transformation protocols are essential skills for any molecular biologist.

• 3. Mini-prep

  Mini-prep, also known as small-scale plasmid DNA extraction, is a widely used technique in molecular biology for the rapid isolation of plasmid DNA from bacterial cultures. This method allows researchers to quickly and efficiently obtain small quantities of plasmid DNA for various downstream applications, such as restriction enzyme analysis, sequencing, or transfection into eukaryotic cells. The mini-prep protocol typically involves the lysis of bacterial cells, followed by the selective precipitation and purification of plasmid DNA, while removing genomic DNA, RNA, and other cellular contaminants. The resulting plasmid DNA is then eluted in a small volume of buffer, ready for further use. Mini-prep is a valuable tool for screening and verifying the successful cloning of DNA fragments into plasmid vectors, as well as for preparing samples for more extensive plasmid purification methods, such as midi-prep or maxi-prep. The simplicity, speed, and cost-effectiveness of mini-prep make it an indispensable technique in the everyday workflow of molecular biology laboratories, enabling researchers to quickly and efficiently obtain high-quality plasmid DNA for a wide range of experimental applications.

• 4. Enzyme cut

  Enzyme cutting, also known as restriction enzyme digestion, is a fundamental technique in molecular biology that involves the use of specialized enzymes to cleave DNA molecules at specific nucleotide sequences. Restriction enzymes, also called restriction endonucleases, recognize and bind to short, palindromic DNA sequences, known as restriction sites, and then catalyze the hydrolysis of the phosphodiester bonds within the DNA, resulting in the generation of discrete DNA fragments. This process is essential for a wide range of molecular biology applications, including the construction of recombinant DNA molecules, the analysis of DNA structure and sequence, and the preparation of DNA samples for downstream techniques such as cloning, sequencing, and Southern blotting. The ability to precisely control the fragmentation of DNA using restriction enzymes has been a crucial tool in the development of modern molecular biology and has enabled significant advancements in our understanding of genetic information and the manipulation of genetic material. As such, the mastery of enzyme cutting techniques is a fundamental skill for any researcher working in the field of molecular biology.