2. 플라즈미드 DNA vector.

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1 2. 플라즈미드 DNA vector

2 플라즈미드(Plasmid); small circular double-stranded DNA molecules 대장균에서 증식
구성 요소 Ori; origin of replication in E. coli Cloning site; 제한 효소 Antibiotic resistance marker gene 필요한 것 제한 효소(restriction endonuclease enzyme) ; 가위 Ligase ; 풀 해당 DNA Competent E. coli cells Selection media EcoRI EcoRI EcoRI Kana Amp Kana EcoRI EcoRI

3 Bacterial Transformation with a Plasmid
E. coli cell Amps chromosome 실험실에서 competent 상태(DNA를 잘 흡수할 수 있는)로 준비함 Ampicillin 감수성 세균 Transform with Ampr plasmid Ampr Ampicillin 저항성 세균 E. coli cell Ampr + plasmid

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5 박테리아 배양 Luria-Bertani medium Tryptone 10 g/l, Yeast extract 5 g/l, NaCl 10 g/l 조건 37 ºC, rpm 유산소 회전 밀도 2-3 x 109 세포/ml 1 OD= 0.8 x 109 세포/ml

6 DNA 농도 측정 자외선 흡수 분광기(ultraviolet absorbance spectrophotometry)
260 nm; 1= 50 μg/ml 흡광도 비 A260/A 이상

7 플라즈미드 DNA 분리 세포배양 (37ºC, shaking overnight) 세포 침전 ; Rnase 처리 세포 용해 (lysis) ; NaOH, SDS 중화 (neutralization); KOAcet 분리 ; Phenol 혹은 칼럼으로 플라즈미드 분리 이 과정에서 genomic DNA, cell 찌꺼기, 단백질 제거 정제 ; EtOH 침전이나 칼럼으로 순수 플라즈미드 정제 농도 검정 ; uv spectrophotometer (260 nm) – 석영 큐벳 1 O.D. = 50 μg/ml double-stranded DNA 38 μg/ml single-stranded DNA or RNA 전기영동 ; DNA 크기 상태 확인

8 플라즈미드 DNA 분리 원리 및 방법 Cell pellet (1-5 ml overnight culture) Resuspension Sol I; Glucose is added to increase the osmotic pressure outside the cells. Tris is a buffering agent used to maintain a constant pH ( = 8.0). EDTA protects the DNA from degradative enzymes (called DNAses); EDTA binds divalent cations that are necessary for DNAse activity. 3. Lysis Sol II; The alkaline mixtures ruptures the cells, and the detergent breaks apart the lipid membrane and solubilizes cellular proteins. NaOH also denatures the DNA into single strands. 4. Neutralization Sol III: The acetic acid neutralizes the pH, allowing the DNA strands to renature. The potassium acetate also precipitates the SDS from solution, along with the cellular debris. The E. coli chromosomal DNA, a partially renatured tangle at this step, is also trapped in the precipitate. The plasmid DNA remains in solution. 5, Centrifuge tubes for 5 minutes at high speed. 6. Take supernatant and fill remainder of centrifuge tube with isopropanol. Isopropanol effectively precipitates nucleic acids, but is much less effective with proteins. A quick precipitation can therefore purify DNA from protein contaminants. 7. Centrifuge tubes for 5 minutes. 8. Add ice-cold 70% ethanol and spin tubes for 1 minute. 9. Pour off supernatant (be careful not to dump out pellet) and drain tube on paper towel. Ethanol helps to remove the remaining salts and SDS from the preparation. Allow tube to dry for ~5 minutes. Add 50 ul TE to tube. If needed, centrifuge tube briefly to pool TE at bottom of tube. DNA is ready for use and can be stored indefinitely in the freezer. Solutions: Solution 1: 50 mM glucose 25 mM Tris-HCl pH mM EDTA pH 8.0 Solution 2: 1% SDS 0.2 N NaOH Solution 3: 3 M Potassium Acetate TE:10 mM Tris-HCl pH mM EDTA pH 8.0

9 DNA 정제 단백질 제거; 페놀 혹은 페놀/클로로포름 1/1 혼합물 DNA 침전; 에탄올 혹은 아이소프로파놀

10 CTAB(Cetyltrimetylammonium bromide)
식물 DNA 추출 핵산-CTAB 복합체가 침전되고 탄수화물, 단백질 같은 오염물은 상등액 1M NaCl 용액에 녹이면 복합체가 떨어짐

11 투명 용해질의 제법 알칼리 변성

12 EtBr(ethidium bromide)
밀도기울기 원심분리법(density gradient centrifugation) DNA (buoyant density) 부력 밀도 1.7g/cm3 UV 쪼임 순수한 플라즈미드 DNA 얻는데 매우 유용

13 절단 초나선형 열린 원형