A+ 생화학실험 <6주차. Plasmid DNA Miniprep> 레포트

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1. Plasmid DNA

Plasmid DNA는 박테리아 내에 존재하는 extrachromosomal DNA로, 자체적 복제가 가능한 복제 기점을 가지고 있습니다. 생존에 필수적이지 않은 정보를 담고 있으며, 항생제 내성 유전자 같은 선택적 마커가 포함되기도 합니다. 이를 통해 항생제가 포함된 배지에서 cell-selection을 진행할 수 있으며, 이로부터 유전자 발현 여부를 조사할 수 있습니다. 또한 plasmid DNA는 super coiled 형태의 DNA로, 유전자 조작, 분리, cloning 등 유전공학 분야에 용이하게 활용됩니다.
2. Plasmid DNA extraction

Plasmid DNA extraction은 세포 내에 존재하는 작고 원형의 DNA 분자인 plasmid DNA를 분리하고 정제하기 위한 일련의 실험 과정을 의미합니다. 일반적으로 세포 벽을 분해하고 세포 내용물을 분리한 다음, 화학적 혹은 물리적 방법을 통하여 plasmid DNA를 순수하게 추출하는 단계를 포함합니다. 본 실험에서는 alkaline lysis method를 사용하여 plasmid DNA를 추출하였습니다.
3. Alkaline lysis method

Alkaline lysis method는 높은 pH 환경에서 세포를 처리한 후 중화하여 plasmid DNA만을 추출하는 방법입니다. 먼저, 높은 pH에서 세포를 파괴하여 DNA를 방출시킨 뒤, 낮은 pH로 중화시켜 genomic DNA와 단백질을 침전시키고, 상대적으로 안정한 plasmid DNA를 용액 상층부에서 회수합니다. 이 방법은 빠른 처리 속도 및 높은 수율이라는 장점을 가지고 있습니다.
4. Resuspension, Lysis, Neutralization

Plasmid DNA 추출 과정은 세 단계로 구성됩니다. 첫 번째 단계인 Resuspension 단계에서는 세포를 안정적으로 처리하고 효율적인 DNA 정제를 위한 기반을 마련합니다. 두 번째 단계인 Lysis 단계에서는 높은 pH와 계면활성제를 이용하여 세포를 파괴하고 DNA를 방출시킵니다. 마지막 Neutralization 단계에서는 pH를 중성으로 조절하여 plasmid DNA와 genomic DNA를 분리합니다.
5. Purification of plasmid DNA

Alkaline lysis 후 추출된 plasmid DNA는 spin column을 이용한 정제 과정을 거칩니다. 이 과정에서 silica membrane에 DNA가 결합하고, 세척 및 용출 단계를 통해 순도 높은 plasmid DNA를 얻을 수 있습니다. 특히 elution buffer의 낮은 이온 강도와 높은 pH 조건에서 DNA와 silica 사이의 결합이 약화되어 DNA가 용출됩니다.
6. Agarose gel electrophoresis

추출한 plasmid DNA의 크기와 순도를 확인하기 위해 agarose gel 전기영동을 수행하였습니다. 전기영동 결과, 추출한 DNA의 크기가 약 5007-5991 bp 범위인 것으로 확인되었으며, 단일 진한 band가 관찰되어 높은 순도의 plasmid DNA를 얻었음을 알 수 있었습니다.
7. DNA concentration measurement

추출한 plasmid DNA의 농도를 UV/VIS 분광광도계로 측정한 결과, 152 ng/μL로 나타났습니다. 또한 260/280 비율이 2.09로 측정되어 순도가 높은 DNA를 얻었음을 확인하였습니다.

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1. Plasmid DNA

Plasmid DNA is a circular, double-stranded DNA molecule that exists independently of the chromosomal DNA in bacterial cells. Plasmids are widely used in molecular biology and biotechnology as vectors for gene cloning, expression, and manipulation. They offer several advantages, such as their small size, ease of manipulation, and ability to replicate independently within the host cell. Plasmids can carry genes of interest and be introduced into host cells, allowing for the production of desired proteins or the study of gene function. Understanding the properties and applications of plasmid DNA is crucial for various research and industrial applications, including genetic engineering, protein production, and vaccine development.
2. Plasmid DNA extraction

Plasmid DNA extraction is a fundamental technique in molecular biology and biotechnology. It involves the isolation and purification of plasmid DNA from bacterial cells, which is essential for various downstream applications, such as gene cloning, DNA sequencing, and genetic engineering. The process typically involves cell lysis, separation of plasmid DNA from chromosomal DNA and other cellular components, and finally, the purification of the plasmid DNA. Several methods, such as alkaline lysis, column-based purification, and magnetic bead-based extraction, are commonly used to extract plasmid DNA efficiently and with high purity. The choice of extraction method depends on factors like the size and copy number of the plasmid, the desired yield and purity, and the specific requirements of the downstream application. Effective plasmid DNA extraction is crucial for ensuring the success of various molecular biology experiments and applications.
3. Alkaline lysis method

The alkaline lysis method is a widely used technique for the extraction of plasmid DNA from bacterial cells. This method relies on the differential denaturation of chromosomal DNA and plasmid DNA under alkaline conditions. The process involves three main steps: resuspension, lysis, and neutralization. During the resuspension step, the bacterial cells are resuspended in a buffer containing glucose, Tris, and EDTA, which helps to maintain the integrity of the cells. In the lysis step, the cells are treated with a solution containing sodium hydroxide and SDS, which disrupts the cell membrane and denatures the chromosomal DNA. The final neutralization step involves the addition of a high-salt solution, typically potassium acetate, which causes the chromosomal DNA and cellular debris to precipitate, while the plasmid DNA remains in solution. The alkaline lysis method is relatively simple, cost-effective, and can yield high-quality plasmid DNA suitable for various downstream applications, making it a widely adopted technique in molecular biology laboratories.
4. Resuspension, Lysis, Neutralization

The three key steps in the alkaline lysis method for plasmid DNA extraction are resuspension, lysis, and neutralization. The resuspension step involves suspending the bacterial cells in a buffer containing glucose, Tris, and EDTA, which helps to maintain the integrity of the cells and prepare them for lysis. The lysis step uses a solution containing sodium hydroxide and SDS to disrupt the cell membrane and denature the chromosomal DNA. The final neutralization step involves the addition of a high-salt solution, typically potassium acetate, which causes the chromosomal DNA and cellular debris to precipitate, while the plasmid DNA remains in solution. This step is crucial for separating the plasmid DNA from the chromosomal DNA and other cellular components, allowing for the efficient purification of the plasmid DNA. The careful execution of these three steps is essential for obtaining high-quality plasmid DNA with minimal contamination, which is crucial for downstream applications in molecular biology and biotechnology.
5. Purification of plasmid DNA

The purification of plasmid DNA is a critical step in the extraction process, as it ensures the removal of contaminants and the recovery of high-quality plasmid DNA suitable for various applications. After the initial steps of resuspension, lysis, and neutralization, the plasmid DNA needs to be further purified to remove any remaining cellular debris, proteins, RNA, and other impurities. Common purification methods include column-based chromatography, where the plasmid DNA is selectively bound to a resin and then eluted, and precipitation-based techniques, such as the use of isopropanol or ethanol. These purification methods take advantage of the physical and chemical properties of plasmid DNA to separate it from other cellular components. The choice of purification method depends on factors such as the desired purity, yield, and downstream application requirements. Effective purification is crucial for ensuring the integrity and functionality of the plasmid DNA, which is essential for applications in genetic engineering, molecular biology research, and biotechnology.
6. Agarose gel electrophoresis

Agarose gel electrophoresis is a widely used analytical technique in molecular biology for the separation and visualization of DNA molecules, including plasmid DNA. This method relies on the principle that DNA molecules migrate through an agarose gel matrix under the influence of an electric field, with the rate of migration being inversely proportional to the size of the DNA fragments. Agarose gel electrophoresis allows for the separation of DNA molecules based on their size, enabling the identification and analysis of plasmid DNA samples. This technique is commonly used to verify the size and purity of plasmid DNA, as well as to assess the success of various DNA manipulation and cloning experiments. The visualization of the separated DNA bands is typically achieved through the use of fluorescent dyes, such as ethidium bromide or SYBR Green, which intercalate with the DNA and emit light when exposed to UV radiation. Agarose gel electrophoresis is a crucial tool in the field of molecular biology, providing valuable information about the quality and characteristics of plasmid DNA samples.
7. DNA concentration measurement

Accurate measurement of DNA concentration, including plasmid DNA, is essential for various applications in molecular biology and biotechnology. Several methods are available for determining the concentration of DNA samples, with the most common being spectrophotometric analysis. This technique relies on the ability of DNA to absorb ultraviolet (UV) light, with the absorbance at a specific wavelength (typically 260 nm) being directly proportional to the DNA concentration. Other methods, such as fluorometric assays and digital PCR, can also be used to quantify DNA concentration with high sensitivity and accuracy. The choice of method depends on factors like the sample volume, the presence of contaminants, and the required level of precision. Accurate DNA concentration measurement is crucial for ensuring the proper normalization and standardization of DNA samples, which is essential for downstream applications such as cloning, sequencing, and gene expression analysis. Proper quantification of plasmid DNA is particularly important for applications like gene delivery, protein production, and genetic engineering, where the precise amount of plasmid DNA is a critical parameter.

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2012.09.07· 6페이지