1. Preparation of Midi-Scale Plasmid DNA from E
Preparation of Midi-Scale Plasmid DNA from E. coli (midi-scale plasmid DNA)
생화학 실험 (2)
1주차
담당교수 : 송재환 교수님
담당조교 : 오대석, 이민식

2. Contents Plasmid DNA for gene cloning Components of plasmid DNA
Plasmid purification methods
Preparation of Plasmid DNA
Identification of purified plasmid DNA

3. 1. Plasmid DNA for gene cloning
Principle of gene cloning
1. plasmid DNA replication
2. cell proliferation
colony
8~12h Incubation

4. 2. Components of plasmid DNA
Plasmid DNA component
Origin of replication
2. Selectable marker (Antibiotic Resistance gene)
3. Multiple cloning site
MC4R TM2+3 (pET32a) Cloning
E N L Y F Q G S P Met Y F F I C S L A V A D Met L V S V S N G S E T I V I T L L N S T D T D A Q S F T V N I D N V I D S V I C S S L L A S I C S L L S I A V = 76개
-> 76개 아미노산 = 228bp
-> Total bp: bp = 6128 bp

5. 3. Plasmid purification methods
Alkaline lysis Method
2. Anion-Exchange Chromatography Method
Cell lysis
DNA separation
Types of preperation
DNA yield(ug)
Mini
100ug>
Midi
Maxi
Mega
Giga
purification scale

6. Alkaline cell lysis Solution II – Lysis solution
Solution I – Resuspension solution
50mM glucose = 삼투압을 유지시켜 세포의 외형을 유지시켜 준다.
25mM Tris-Cl pH 8.0 = ph를 맞춰줌
10mM EDTA, pH8.0 = 세포벽의 integrity를 유지하는데 필수적인 calcium ion을 제거하여 세포
벽이 군데군데 무너지게 만듬
Solution II – Lysis solution
0.2N NaOH = DNA가 세포가 깨지면서 밖으로 나왔을 때 DNA을 변성시키는 역할을 한다(염기성).
1%(W/V) SDS = 계면 활성제로 cell을 lysis 시킴
Solution III – Neuralization solution
5M Potassium acetate,
Glacial acetic acid = 강력한 산으로 위에서 NaOH에 의해 염기성이 된 용액
을 다시 중성으로 돌려 놓음으로써 DNA 를 재결합시키는 역할을 한다.

7. Alkaline lysis Method

8. Anion-Exchange Chromatography Method
Buffer EQU (low salt): DNA binding
Buffer WASH (medium salt): RNA, protein, carbohydrate등의 제거가능
Buffer ELU (high salt): Salt가 DNA binding을 억제하여 elution이 가능

9. 4. Preparation of Plasmid DNA

10. Materials Alkaline cell lysis Anion-exchange-based DNA purification
Solution I – Resuspension solution
50mM glucose
25mM Tris-Cl pH 8.0
10mM EDTA, pH8.0
Solution II – Lysis solution
0.2N NaOH
1%(W/V) SDS
Solution III – Neuralization solution
5M Potassium acetate
Glacial acetic acid
Anion-exchange-based DNA purification
Anion-exchange-based column
Buffer EQU buffer-low salt
Buffer WASH buffer-medium salt
Buffer ELU buffer-high salt
isopropanol
70% Et-OH
D.W

11. Alkaline cell lysis method
Procedures
1) Resuspension the pellet in 3 ml of Alkaline lysis solution I (Buffer RES)
“ Vol. [ mL ] = Culture Volume [ mL ] x OD600 / 50”
- Culture volume: 100ml , O.D600: 1.5 => 3ml
2) Add 3ml solution II (Buffer LYS)
- Mix the contents thoroghly by inverting the tube several times(5 times).
- Incubate the mixture at room temperature (18–25 °C) for 5 min.
3) Column equilibration (Buffer EQU) 12ml
4) Add 3 ml solution III (Buffer NEU)
- inverting the tube several times(10 ~15 times)
5) Inverting the tube 3 times directly before applying the lysate to the equilibrate NucleoBond® Xtra Column Filter
6) Wash column filter and column (Buffer EQU) 5ml
7) Discard column filter

12. Anion-exchange chromatography method
7) Add 8ml of Buffer WASH into column and allow the column to empty by gravity flow 8) Elution - Add 3ml (Buffer ELU) 9) Add 3ml isopropanol 10) Centrifugate for 30min at 13000rpm at 4˚C in a microcentrifuge 11) Carefully decant the supernatant. 12) Wash with 2ml of 70% Et-OH 13) Centrifugate for 5min at 13000rpm, RT in a microcentrifuge 14) Carefully decant the 70% Et-OH 15) Air dry 5min and redissolve the DNA in 400ul D.W.

13. 5. Identification of purified plasmid DNA

14. Result
생화학과 홈페이지 자료실 upload

15. Report
마감기한: 2012/9/7 5시 제출장소: s403 (구조생화학 및 분자생물리학 연구실) 보고서 형식엄수(생화학과 홈페이지-자료실-조교) 자필작성만 점수부여
결과 분석시 고려사항
plasmid DNA 농도-Total plasmid DNA 양
A260/A280 및 A260/230 ratio의 의미 및 결과분석
plasmid DNA 전기영동 결과분석