2 3 Restriction Mapping of Circular Plasmid DNA

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생화학 실험 (2) 3주차 Restriction Mapping of Circular Plasmid DNA and Transformation of Ligated

생화학 실험 (2) 3주차 Restriction Mapping of Circular Plasmid DNA and Transformation of Ligated Plasmid DNA 담당교수 : 하 상 준 교수님 담당조교 : 홍 석 봉 조 교 이수영조 교

1. Transformation of ligated plasmid DNA 2. Restriction Mapping of circular plasmid DNA

1. Transformation of ligated plasmid DNA 2. Restriction Mapping of circular plasmid DNA

Transformation 이번 주 실험 Vector transformation 지난 주 실험 Colony형성

Transformation 이번 주 실험 Vector transformation 지난 주 실험 Colony형성

Transformation

Transformation

Materials 1. Ligated plasmid DNA, D. W. (negative control) 2. competent cell 3. 200

Materials 1. Ligated plasmid DNA, D. W. (negative control) 2. competent cell 3. 200 ul micropipette LB/AMP plate : 4. spreader yeast extract 5. alcohol lamp Tryptone 6. 42 ℃ Heat block 7. LB-Amp plates 8. ice box & ice Na. Cl Agar Ampicillin (antibiotics)

Procedure 1. Competent cell을 ice 속에서 녹임 (2 -3 min) 2. Ligated DNA 10

Procedure 1. Competent cell을 ice 속에서 녹임 (2 -3 min) 2. Ligated DNA 10 ul와 negative control 10 ul을 competent cell에 넣는다. 3. Ice 속에서 30분간 방치 4. 42 ℃ Heat block에 samples을 넣고 90초 동안 heat-shock 5. Ice 속에서 2분간 방치 6. LB 배지 200 ul를 넣고, 37℃에서 15분간 incubation 7. Spreading 8. 37 ℃ incubator에서 plates를 뒤집어 colonies가 형성될 때까지 배양 (약 12~18시간)

Restriction Mapping of circular plasmid DNA Restriction Enzyme : Eco. R I, Hind III,

Restriction Mapping of circular plasmid DNA Restriction Enzyme : Eco. R I, Hind III, Xho I ? Eco. R I 4000 bp Hind III 4000 bp Eco. R I + Hind III Eco. R I + Xho I Hind III Xho I 1000 bp + 3000 bp 1500 bp + 2500 bp + 3500 bp Eco. R I 4 kb Hind III Xho I

Materials 1. Unknown plasmid DNA 2. micropipette 3. Microcentrifuge tube (1. 5 ml) 4.

Materials 1. Unknown plasmid DNA 2. micropipette 3. Microcentrifuge tube (1. 5 ml) 4. (10 x) restriction enzyme buffer 5. Restriction enzyme ( Xho I, Eco. R V, Pst I ) → -20℃ 6. 37 ℃ water bath 7. (6 X) agarose loading dye 8. DNA size marker 9. ice box and ice

Procedure 1. Microcentrifuge tube에 mapping할 unknown DNA와 buffer, D. W를 다음과 같이 첨가하여 반응

Procedure 1. Microcentrifuge tube에 mapping할 unknown DNA와 buffer, D. W를 다음과 같이 첨가하여 반응 혼합물을 만든다. (total volume : 10 ul) ① Eco. R V ② Xho I ③ Eco. R V+Xho I ④ Xho I+Pst I ⑤ Eco. R V+Pst I Eco. R V Xho I Pst I Eco. R V Pst I Unknown DNA ( 0. 1 ug/ul) 2 2 2 Enzyme buffer (10 X) 1 1 1 Enzyme은 제일 마지막에 넣어준다. -> 1 Restriction enzymes D. W. 6 6 5 5 5

Procedure 2. 37 ℃에서 1시간 동안 reaction한다. 3. Enzyme reaction한 DNA fragment들에 (6 x)agarose

Procedure 2. 37 ℃에서 1시간 동안 reaction한다. 3. Enzyme reaction한 DNA fragment들에 (6 x)agarose dye 2 ul 넣고 agarose gel electrophoresis를 수행한다. reaction volume (10 ul) + agrose dye (2 ul) = total 12 ul (DNA size marker도 loading한다. ) # Restriction enzyme 다룰 때의 주의사항 ! -Enzyme이 들어있는 tube의 아랫부분을 손으로 만지지 않는다. -enzyme의 양은 total volume에 10%를 넘지 않는다. -Tip 윗부분만 살짝 넣어서 enzyme을 딴다.

Result # Restriction mapping 1. restriction enzyme digestion한 DNA의 gel electrophoresis 사진 2. restriction

Result # Restriction mapping 1. restriction enzyme digestion한 DNA의 gel electrophoresis 사진 2. restriction map # Transformation 1. transformation 사진

Further Study DNA를 세포 내에 주입할 때 쓰이는 용어들로 transformation, transfection, transduction이 있다. 이들의

Further Study DNA를 세포 내에 주입할 때 쓰이는 용어들로 transformation, transfection, transduction이 있다. 이들의 차이점은 무엇일까?

2 4 Restriction Mapping of Circular Plasmid DNA2 4 Restriction Mapping of Circular Plasmid DNA
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DNA Restriction enzyme DNA ligase DNA PCR DNADNA Restriction enzyme DNA ligase DNA PCR DNA
Plasmid u Plasmid Extrachromosomal DNA 2 200 kbPlasmid u Plasmid Extrachromosomal DNA 2 200 kb
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Restriction Enzymes Restriction Enzymes Enzyme that cuts DNARestriction Enzymes Restriction Enzymes Enzyme that cuts DNA
Cutting DNA b Restriction endonucleases restriction enzymes stickyCutting DNA b Restriction endonucleases restriction enzymes sticky
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