[A]서강대학교 현대생물학실험2_2차 풀레포트_TA Cloning과 Mini-Prep, Restriction enzyme & Gel electrophoresis

본 내용은 " [A]서강대학교 현대생물학실험2_2차 풀레포트_TA Cloning과 Mini-Prep, Restriction enzyme & Gel electrophoresis " 의 원문 자료에서 일부 인용된 것입니다. 2024.07.16

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• 1. TA Cloning

  TA Cloning은 PCR을 거친 product의 3'에 존재하는 A overhang과 T vector의 3'의 T overhang의 상보성으로 인해 cloning을 진행하는 기법이다. Lac Z selection을 통해 blue colony와 white colony를 확인하여 insert DNA가 정상적으로 삽입되었는지 확인할 수 있다. Self-ligation을 줄이기 위해 alkaline phosphatase 처리나 T-overhang cloning protocol 수정 등의 방법을 사용할 수 있다.

• 2. Mini-Prep

  Mini-Prep은 Alkaline lysis 기법을 이용하여 plasmid DNA를 추출하는 방법이다. Resuspension, Lysis & denaturation, Neutralization, renaturation, EtOH precipitation 단계를 거쳐 plasmid DNA만 분리할 수 있다. 영양분이 많은 TB(Terrific broth)로 배양하면 plasmid 생산량을 높일 수 있다.

• 3. Restriction Enzyme

  제한효소는 특정 염기서열을 인식하여 DNA를 절단하는 효소이다. 본 실험에서 사용한 HindⅢ와 NdeⅠ는 Type Ⅱ 제한효소로, 인식부위에서 정확하게 절단한다. 각 효소의 최적 buffer 조건이 다르므로, 개별적으로 처리하면 효율을 높일 수 있다.

• 4. Gel Electrophoresis

  전기영동을 통해 plasmid DNA의 크기와 conformation을 확인할 수 있다. Uncut 시료에서는 다양한 band가 관찰되며, 제한효소 처리 시 예상 크기의 band가 나타난다. 작은 DNA 단편은 1% agarose gel에서 관찰하기 어려울 수 있으므로, 농도를 높여 전기영동을 진행해야 한다.

• 5. Beer-Lambert Law

  흡광도와 Beer-Lambert Law를 이용하여 DNA 농도를 계산할 수 있다. 시료를 적절히 희석하여 측정하면 정확한 농도를 구할 수 있다.

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• 1. TA Cloning

  TA cloning is a widely used molecular biology technique that allows for the efficient insertion of PCR-amplified DNA fragments into a vector. This method takes advantage of the non-template-dependent addition of a single adenine (A) nucleotide to the 3' ends of PCR products by certain thermostable DNA polymerases, such as Taq polymerase. The vector used in TA cloning is typically linearized and has a single, complementary thymine (T) overhang at each end, enabling the PCR product to ligate efficiently. TA cloning is a simple, fast, and cost-effective approach that is particularly useful for cloning PCR products without the need for restriction enzyme digestion or the creation of compatible ends. It is commonly employed in various applications, including gene expression, protein production, and the construction of genetic libraries. The ease of use and high efficiency of TA cloning make it a valuable tool in molecular biology research and biotechnology.

• 2. Mini-Prep

  Mini-prep, also known as small-scale plasmid DNA isolation, is a widely used technique in molecular biology for the rapid and efficient extraction of plasmid DNA from bacterial cultures. This method allows researchers to quickly obtain small amounts of purified plasmid DNA, which can then be used 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, the removal of cellular debris and chromosomal DNA, and the selective precipitation or binding of plasmid DNA. The simplicity and speed of the mini-prep procedure make it an indispensable tool in molecular biology laboratories, enabling researchers to quickly screen and analyze multiple plasmid constructs. Additionally, the small-scale nature of the mini-prep allows for the efficient processing of numerous samples simultaneously, making it a highly versatile and time-saving technique.

• 3. Restriction Enzyme

  Restriction enzymes are essential tools in molecular biology, playing a crucial role in various genetic engineering and DNA manipulation techniques. These enzymes are derived from bacteria and archaea and have the ability to recognize and cleave specific DNA sequences, known as restriction sites. Restriction enzymes are classified based on their recognition sequences, cleavage patterns, and cofactor requirements. They are widely used for the precise cutting and manipulation of DNA, enabling the creation of recombinant DNA molecules, the analysis of DNA structure and sequence, and the development of genetic tools such as plasmids and expression vectors. The availability of a diverse array of restriction enzymes with varying specificities allows researchers to tailor their experiments and precisely engineer DNA constructs. The use of restriction enzymes has been instrumental in the advancement of molecular biology, biotechnology, and genetic research, facilitating the study and manipulation of genetic information at the molecular level.

• 4. Gel Electrophoresis

  Gel electrophoresis is a fundamental technique in molecular biology and biochemistry, used for the separation and analysis of DNA, RNA, and proteins based on their size and charge. This technique involves the migration of charged molecules through a porous gel matrix under the influence of an electric field. Depending on the type of gel and the buffer system used, different biomolecules can be effectively separated and visualized. DNA gel electrophoresis, for instance, is commonly used to separate DNA fragments based on their size, allowing researchers to analyze the composition of DNA samples, verify the success of cloning or PCR experiments, and perform DNA fingerprinting. Protein gel electrophoresis, on the other hand, is employed to separate and characterize proteins based on their molecular weight and charge. The versatility and high resolution of gel electrophoresis make it an indispensable tool in various fields, including molecular biology, genetics, biochemistry, and proteomics, enabling the study and manipulation of biological macromolecules with precision and accuracy.

• 5. Beer-Lambert Law

  The Beer-Lambert law, also known as the Beer-Lambert-Bouguer law, is a fundamental principle in spectroscopy that describes the relationship between the absorption of light by a substance and the properties of that substance. This law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species and the path length of the light through the solution. Mathematically, the Beer-Lambert law is expressed as A = ε × c × l, where A is the absorbance, ε is the molar extinction coefficient, c is the concentration of the absorbing species, and l is the path length. This relationship allows for the quantitative analysis of the concentration of a substance in a solution by measuring its absorbance at a specific wavelength. The Beer-Lambert law is widely used in various fields, including analytical chemistry, biochemistry, and spectroscopy, for the determination of analyte concentrations, the characterization of materials, and the study of molecular interactions. Its simplicity and accuracy make it a valuable tool in both research and industrial applications.