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분야
docx2. Date
3. Experiment
3.1. Day 1 - 제한효소를 이용한 DNA 절단 및 분석, Ligation
3.1.1. Digestion of DNA with Restriction Enzyme
I. Apparatus
II. Reagents
3.1.2. Agarose Gel Electrophoresis
I. Apparatus
II. Reagents
III. Principle
IV. Result
3.1.3. Gel Purification
I. Apparatus
II. Reagents
III. Principles
3.1.4. Ligation
I. Reagents
II. Principles
3.2. Day 2 – 분리한 유전자를 competent cell에 삽입
3.2.1. Transformation(형질전환)
I. Apparatus
II. Reagents
III. Principles
V. Result
3.3. Day 3 – transformant 확인 및 subculturing
3.3.1.
I. Apparatus
II. Reagents
III. Principles
3.4. Day 4
3.4.1. Protocol for Plasmid Prep
I. Apparatus
II. Reagents
III. Principles
IV. Result
3.5. Day 5 – polymerase chain reaction
3.5.1. Semi-quantitative-PCR (Polymerase Chain Reaction)
I. Apparatus
II. Reagents
III. Principles
V. Result
4. Overall Observations
4.1. Result
4.2. Discussion
4.2.1. Experiment 1 (Day 1-4)
4.2.2. Experiment 2 (Day 5)
5. Reference
제한효소 활용 및 Ligation, 전기영동, plasmid prep. 등의 여러 실험을 통해 Gene cloning의 전반적인 실험 및 이해를 하고, PCR의 기초적인 간단한 실험을 함으로써 DNA와 유전자를 다루는 molecular biology에서의 이론적 측면뿐만 아니라 실험적인 측면 또한 함양한다.
2. Date
실험기간: 2011년 11월 7일 – 11일, 총 5일
3. Experiment
3.1. Day 1 - 제한효소를 이용한 DNA 절단 및 분석, Ligation
3.1.1. Digestion of DNA with Restriction Enzyme
I. Apparatus
- Water bath, brief centrifuge
II. Reagents
- Plasmid, DDW, restriction enzymes [NdeI, BamHI], restriction enzyme reaction buffer
① NdeI: an endonuclease isolated from Neisseria denitrificans. In molecular biology, it is commonly used as a restriction enzyme.
② BamHI: a restriction enzyme, derived from Bacillus amyloliquefaciens. It has the recognition site (G'GATCC), and leaves a sticky end. One of the earlier enzymes to be used, it is popular for historical reasons, but also because digestion leaves a GATC overhang compatible with many other enzymes. Persistent issues with this specific enzyme spontaneously failing to generate cloneable fragments necessitate aliquoting prior to use. Engineered variants of the enzyme have been created in order to avoid star activity.
