Introduction Sitespecific mutagenesis The cause of mutagenesis The

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Introduction. Site-specific mutagenesis • The cause of mutagenesis. • The types of mutagenesis. 1.

Introduction. Site-specific mutagenesis • The cause of mutagenesis. • The types of mutagenesis. 1. UV. 1. Single base mutation. 2. Chemical-Carcinogen. 2. Multiple mutation. 3. Error prone of PCR. 3. Insertion. 4. Site-directed mutagenesis. 4. Deletion.

뺄것 Overlap Extension PCR 3’ 5’ 5’ 3’ Primer 1 3’ Primer 3 5’

뺄것 Overlap Extension PCR 3’ 5’ 5’ 3’ Primer 1 3’ Primer 3 5’ 5’ 3’ 3’ 5’ Primer 2 5’ 3’ 5’ Primer 4 3’ 1 Cycle 2 Cycle

Overlap Extension PCR Ligation PCR Primer 4 Primer 1 1 Cycle 2 Cycle Primer로써

Overlap Extension PCR Ligation PCR Primer 4 Primer 1 1 Cycle 2 Cycle Primer로써 기능!!

뺄것 541 586 631 676 721 766 811 856 901 946 Overlap Extension PCR

뺄것 541 586 631 676 721 766 811 856 901 946 Overlap Extension PCR ttcacatgccctcagtgccgaaagagctttcctcggcggagcttc cgccccaacctgcagctggccaatatggtccaggtgattcggcag atgcacccaacccctggtcgagggagccgcgtgaccgatcagggc atctgtcccaaacaccaagaagccctgaagctcttctgcgaggta gacgaagaggccatctgtgtgccgagaatccaggagccac aaacagcgtggtgccattggaggaggtggtgcaggagtac aaggccaaactgcaggggcacgtggaaccactgaggaagcacctg gaggcagtgcagaagatgaaagccaaggaggagaggcgagtgaca gaactgaagagccagatgaagtcagagctggcagcggtggcctcg gagtttgggcgactgacacggtttctggctgaagagcaggg

Experimental Procedure : Gel Extraction 1. Agarose gel : membrane binding solution = 10

Experimental Procedure : Gel Extraction 1. Agarose gel : membrane binding solution = 10 mg : 10 ㎕ 씩 넣어 55℃ heat block 에서 10분간 녹인다. 2. 잘 녹았는지를 vortexing 을 통해 확인 후, sample을 column으로 옮긴다. 3. 14000 rpm, 1 min centrifuge 4. Wash buffer 750 ㎕ 를 넣은 후 14000 rpm, 1 min centrifug 5. 한번 더 14000 rpm, 1 min centrifuge하여 남은 wash buffer를 제거한다. 6. Column을 새 tube에 옮긴 후 D. W. 를 30 ㎕ 를 넣고 5분을 기다린다. 7. 14000 rpm, 5 min centrifuge

Experimental Procedure : Ligation PCR • Ligation PCR Component • PCR condition Quantity per

Experimental Procedure : Ligation PCR • Ligation PCR Component • PCR condition Quantity per reaction Targets 1 -10 kb Distilled water 34 ㎕ 5 x Herculase II reaction buffer 10 ㎕ d. NTP mix 0. 5 ㎕ DNA template(1+3) 1㎕ DNA template(2+4) 1㎕ Primer 1+4 2. 5 ㎕ Herculase II fusion DNA polymerase 1㎕ Total reaction volume 50 ㎕ Temp. (℃) Time 95 2 min 95 30 sec 55 30 sec 30 cycles 72 1 min 72 5 min

T-vector cloning. PCR fragment for cloning into T-vector PCR polymerase 의 종류 • Taq

T-vector cloning. PCR fragment for cloning into T-vector PCR polymerase 의 종류 • Taq DNA polymerase • 3’→ 5’ exonuclease 활성 없음 • 특이적으로 3’말단에 A가 생김. • Pfu DNA polymerase • 3’→ 5’ exonuclease 활성 있음 • 3’ 말단에 A가 생기지 않음 < alkaline phosphatase, Calf intestinal(CIP) >

Lac Z 을 이용한 selection Inhibitor Lactose X-Gal Lactose Galactose β-galactosidase + Glucose Blue

Lac Z 을 이용한 selection Inhibitor Lactose X-Gal Lactose Galactose β-galactosidase + Glucose Blue color + Glucose X-gal used to indicate whether a bacterium expresses the β-galactosidase enzyme, which is encoded by the lac Z gene, in a technique called blue/white screening.

Lac Z 을 이용한 selection

Lac Z 을 이용한 selection

Amp/Lac. Z를 이용한 selection

Amp/Lac. Z를 이용한 selection

Experimental Procedur 1. T-vector ligation Ingredient Volume ( ul) 2 x ligation buffer 5

Experimental Procedur 1. T-vector ligation Ingredient Volume ( ul) 2 x ligation buffer 5 T-vector 0. 5 PCR construct (insert) 3. 5 T 4 DNA ligase 1 Total 10 Ligation time 1. Overnight at 16 ℃ 2. 1 ~ 2 hours at Room temperature ◈ Ligation volume setting ng of vector size of vector ng of insert : size of insert = 1 : 5

Experimental Procedure 2. Transformation Positive control Control insert (542 bp) Self-ligation No insert

Experimental Procedure 2. Transformation Positive control Control insert (542 bp) Self-ligation No insert

Experimental Procedure 3. Bacteria culture 16 hr 4. Plasmid DNA Extraction 5. Cloning confirmation

Experimental Procedure 3. Bacteria culture 16 hr 4. Plasmid DNA Extraction 5. Cloning confirmation using two-cut digestion

Report – 결과 및 고찰 1. Gel extraction solution 의 원리 조사 Membrane Wash

Report – 결과 및 고찰 1. Gel extraction solution 의 원리 조사 Membrane Wash Solution 1. 10 m. M potassium acetate (p. H 5. 0) 2. 80% ethanol 3. 16. 7μM EDTA (p. H 8. 0) Membrane Binding Solution 1. 4. 5 M guanidine isothiocyanate 2. 0. 5 M potassium acetate (p. H 5. 0) 2. Lac operon원리와 selection의 원리에 대해서 쓰시오. (원리) 3. 수업자료에 있는 PCR의 2번 째 cycle 에서 합성되는 DNA를 그려보세요. 4. PCR-mediated PCR primer design (Red blank 인 부분을 deletion하는 construct, primer 4개 design 할 것) 5. Design한 primer의 Tm 값과 annealing temperature를 구하시오. (Tm-5℃)

Report - 결과 및 고찰 1 atggctgccgttgccatgacacccaaccctgtgcagacccttcag 46 gaggaggcggtgtgcgccatctgcctcgattacttcacggacccc 91 gtgtccatcggctgcgggcacaacttctgccgagtttgtgtaacc 136 cagttgtggggaggatgaggaggacagagatgagttagat 181

Report - 결과 및 고찰 1 atggctgccgttgccatgacacccaaccctgtgcagacccttcag 46 gaggaggcggtgtgcgccatctgcctcgattacttcacggacccc 91 gtgtccatcggctgcgggcacaacttctgccgagtttgtgtaacc 136 cagttgtggggaggatgaggaggacagagatgagttagat 181 cgggaggaggacggagaggaggaggaagtggaggct 226 gtgggggctggcgcggggtgggacacccccatgcgggatgaagac 271 tacgagggtgacatggaggaggaggtcgaggaggaagaagagggt 316 gtgttctggaccagtggcatgagcaggtccagctgggacaacatg 361 gactatgtgtgggaggacgaggaggaagacctggactac 406 tacttgggggacatggaggacctgaggggggaggatgag 451 gaggacgaggaggaagtgctggaggaggttgaggaagaggatcta 496 gaccccgtcaccccactgcccccgcctccagcccctcggaggtgc 541 ttcacatgccctcagtgccgaaagagctttcctcggcggagcttc 586 cgccccaacctgcagctggccaatatggtccaggtgattcggcag 631 atgcacccaacccctggtcgagggagccgcgtgaccgatcagggc 676 atctgtcccaaacaccaagaagccctgaagctcttctgcgaggta 721 gacgaagaggccatctgtgtgccgagaatccaggagccac 766 aaacagcgtggtgccattggaggaggtggtgcaggagtac 811 aaggccaaactgcaggggcacgtggaaccactgaggaagcacctg 856 gaggcagtgcagaagatgaaagccaaggaggagaggcgagtgaca 901 gaactgaagagccagatgaagtcagagctggcagcggtggcctcg 946 gagtttgggcgactgacacggtttctggctgaagagcaggg 991 ctggaacggcgtctcagagagatgcatgaagcccagctggggcgt 1036 gcgggagccgcggctagtcgccttgcagaacaggccgcccagctc 1081 agccgcctgctggcagaggcccaggagccagcaggggggt 1126 ctccggctgctccaggacatcaaggagactttcaataggtgtgaa