Mini-prep, Restriction Enzyme 분자생물학실험 SUBJECT. Sequence blast Restriction enzyme Mini-prep E.coli transformation TA Ligation PCR DNA EXTRACTION 분자생물학실험.

Presentation on theme: "Mini-prep, Restriction Enzyme 분자생물학실험 SUBJECT. Sequence blast Restriction enzyme Mini-prep E.coli transformation TA Ligation PCR DNA EXTRACTION 분자생물학실험."— Presentation transcript:

1 Mini-prep, Restriction Enzyme 분자생물학실험 SUBJECT

2 Sequence blast Restriction enzyme Mini-prep E.coli transformation TA Ligation PCR DNA EXTRACTION 분자생물학실험

3 3.0Kb → 1.0Kb → 1596bp2076bp Gene2Gene1

4 분자생물학실험

5 E. coli transformation → Selection 분자생물학실험 Materials & Method

6 Blue-white selection of E.coli competent cells using IPTG and X-gal 분자생물학실험 Result & Discussion

7 7 분자생물학실험 Materials & Method Minipreparation (Mini-prep) Reagentconc. Glucose (MW=198.2)50mM Tris-Cl (pH8.0)25mM EDTA (pH8.0)10mM Reagentconc. NaOH0.2N SDS1% Reagentconc. potassium acetate3M acetic acid D.W. Alkaline lysis solution Ⅲ store at 4 ℃ and transfer it to an ice just before use. Alkaline lysis solution Ⅰ autoclave for 15min and store at 4 ℃ Alkaline lysis solution Ⅱ 0.2N NaOH (freshly diluted from a 10N stock) Prepare Sol2 fresh and use at room temperature.

8 8 분자생물학실험 Materials & Method Minipreparation (Mini-prep) Traditional Method 1. Inoculate the single colony into 5ml of LB medium containing the antibiotic and incubate the culture overnight at 37 ℃ with vigorous shaking. 2. Centrifuge at 4000rpm for 5min 3. Remove the medium. 4. Re-suspend the pellet in 100ul Sol Ⅰ by vigorous vortexing. 5. Transfer the re-suspension to new e-tube. 6. Add 200ul Sol Ⅱ and inverting the tube rapidly five times. 7. Rapidly Add 150ul of Sol Ⅲ and inverting the tube several times. 8. Store the tube on ice for 3-5min. 9. Centrifuge at 13,000rpm for 5min at 4 ℃ ( 실험에선 25 ℃ ) 10. Transfer the supernatant into new e-tube and precipitate by 2 volumes of ethanol. 11. Centrifuge at 13,000rpm for 5min at 4 ℃ ( 실험에선 25 ℃ ) 12. Remove the supernatant and add 70% Ethanol. 13. Centrifuge at 13,000rpm for 2min at 4 ℃ ( 실험에선 25 ℃ ) 14. Dry the DNA pellet 15. Dissolve DNA in D.W. with 20ug/ml RNase A

9 9 분자생물학실험 Materials & Method Restriction Enzyme insert pTOP TA V2 R F

10 294bp295bp Lac. ORF 시작

11 분자생물학실험 Materials & Method 10x Buffer 1㎕1㎕ Enzyme (1) 0.2(~0.3) ㎕ Enzyme (2) 0.2(~0.3) ㎕ 10x BSA(1/10 ) 1㎕1㎕ Template 5㎕5㎕ DW Up to 10 ㎕ Total10 ㎕ Restriction enzyme (mixture) PCR tube 에 위의 mixture 넣고 37 ℃ chamber 에 1~2hr 보관 Enzyme 2 종류사용 Enzyme 1 종류 10x Buffer 1㎕1㎕ Enzyme 0.5 ㎕ 10x BSA(1/10 ) 1㎕1㎕ Template 5㎕5㎕ DW Up to 10 ㎕ Total10 ㎕

12 분자생물학실험 Materials & Method 10x Buffer IV 1㎕1㎕ Enzyme (Nco I) 0.2(~0.3) ㎕ Enzyme (Xho I) 0.2(~0.3) ㎕ 10x BSA(1/10) 1㎕1㎕ Template 5㎕5㎕ DW Up to 10 ㎕ Total10 ㎕ Restriction enzyme (mixture) PCR tube 에 위의 mixture 넣고 37 ℃ chamber 에 1~2hr 보관 Enzyme 2 종류사용 Enzyme 1 종류 10x Buffer II 1㎕1㎕ Enzyme(Sac I) 0.2(~0.3) ㎕ 10x BSA(1/10) 1㎕1㎕ Template 5㎕5㎕ DW Up to 10 ㎕ Total10 ㎕ Gene1 Control 10x Buffer 1㎕1㎕ Enzyme(EcoR I) 0.5 ㎕ 10x BSA(1/10) 1㎕1㎕ Template 5㎕5㎕ DW Up to 10 ㎕ Total10 ㎕

13 pTOP TA V2 3807 base pairs agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaa ttaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacgccaagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattcgcccttaagggcgaattctgcagatatccatcacactg gcggccgctcgagcatgcatctagagggcccaattcgccctatagtgaatcgtattacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgtta cccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctatacgtacgg cagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgccggggcgacggatggtgatccccctggccag tgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctcc gttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaatgtcaggcatgagattatcaaa aaggatcttcacctagatccttttcacgtagaaagccagtccgcagaaacggtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgca agcgcaaagagaaagcaggtagcttgcagtgggcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgcc ctctggtaaggttgggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagctctgatcaagagacaggatgaggat cgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgc cgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggct ggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctc accttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcg agcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcg agcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgg gtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctccc gattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcatttt gccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagat ccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcg ccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatg agtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttggg aaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactc tagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagcc ggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagccaggcaactatggatgaacg aaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaag gatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagat cctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggctt cagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttacc agtggctgctgccagtggcgataagtcgtgtcttaccgggttggattcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacac agcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatc cggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgt cgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgt tatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgaatcagtgagcgaggaagcgga ag

14 Gene2 (2076bp) ATGGCGGAATCCGGCGATTTCAACGGTGGTCAACCTCCTCCTCATAGTCCTCTGAGAACAACTTCTTCCG GTAGTAGCAGCAGCAACAACCGTGGTCCTCCTCCTCCTCCTCCTCCTCCTTTAGTGATGGTGAGAAAAAG ATTAGCTTCCGAGATGTCTTCTAACCCTGACTACAACAACTCCTCTCGTCCTCCTCGCCGTGTCTCTCACC TTCTTGACTCCAACTACAATACTGTCACACCACAACAACCACCGTCTCTTACGGCGGCGGCTACTGTATCT TCTCAACCAAACCCACCACTCTCTGTTTGTGGCTTCTCTGGTCTTCCCGTTTTTCCTTCAGACCGTGGTG GTCGGAATGTTATGATGTCCGTACAACCAATGGATCAAGACTCTTCATCTTCTTCTGCTTCACCTACTGTAT GGGTTGACGCCATTATCAGAGACCTTATCCATTCCTCAACTTCAGTCTCTATTCCTCAACTTATCCAAAACG TTAGAGACATTATCTTCCCTTGTAACCCAAATCTCGGTGCTCTTCTTGAATACAGGCTCCGATCTCTCATGC TCCTTGATCCTTCCTCTTCCTCTGACCCTTCTCCTCAAACTTTCGAACCTCTCTATCAGATCTCCAACAATC CTTCTCCTCCACAACAGCAACAGCAGCACCAACAACAACAACAACAGCATAAGCCTCCTCCTCCTCCGAT TCAGCAGCAAGAAAGAGAAAATTCTTCTACCGATGCACCACCGCAACCAGAGACAGTGACGGCCACTGT TCCCGCCGTCCAAACAAATACGGCGGAGGCTTTAAGAGAGAGGAAGGAAGAGATTAAGAGGCAGAAGC AAGACGAAGAAGGATTACACCTTCTCACATTGCTGCTACAGTGTGCTGAAGCTGTCTCTGCTGATAATCTC GAAGAAGCAAACAAGCTTCTTCTTGAGATCTCTCAGTTATCAACTCCTTACGGGACCTCAGCGCAGAGAG TAGCTGCTTACTTCTCGGAAGCTATGTCAGCGAGATTACTCAACTCGTGTCTCGGAATTTACGCGGCTTTG CCTTCACGGTGGATGCCTCAAACGCATAGCTTGAAAATGGTCTCTGCGTTTCAGGTCTTTAATGGGATAAG CCCTTTAGTGAAATTCTCACACTTTACAGCGAATCAGGCGATTCAAGAAGCATTTGAGAAAGAAGACAGT GTACACATCATTGACTTGGACATCATGCAGGGACTTCAATGGCCTGGTTTATTCCACATTCTTGCTTCTAG ACCTGGAGGACCTCCACACGTGCGACTCACGGGACTTGGTACTTCCATGGAAGCTCTTCAGGCTACAGG GAAACGTCTTTCGGATTTCGCAGATAAGCTTGGCCTGCCTTTTGAGTTCTGCCCTTTAGCTGAGAAAGTT GGAAACTTGGACACTGAGAGACTCAATGTGAGGAAAAGGGAAGCTGTGGCTGTTCACTGGCTTCAACAT TCTCTTTATGATGTCACTGGCTCTGATGCACACACTCTCTGGTTACTCCAAAGgtaaaataaacattaccttttaatcact ctttatctataaattattttaagattatataggaaagatatgttctaaaaagtggcttttttggttaatgattggggaatgaacagATTAGCTCCTAAAGT TGTGACAGTAGTGGAACAAGATTTGAGCCACGCTGGTTCTTTCTTAGGAAGATTTGTAGAAGCAATACATT ACTACTCTGCACTCTTTGACTCACTGGGAGCAAGCTACGGCGAAGAGAGTGAAGAGAGACATGTCGTGG AACAGCAGCTATTATCGAAAGAGATACGGAATGTATTAGCGGTTGGAGGACCATCGAGAAGCGGTGAAGT GAAGTTTGAGAGCTGGAGGGAGAAAATGCAACAATGTGGGTTTAAAGGTATATCTTTAGCTGGAAATGCA GCTACACAAGCGACTCTACTGTTGGGAATGTTTCCTTCGGATGGTTACACTTTGGTTGATGATAATGGTAC ACTTAAGCTTGGATGGAAAGATCTTTCGTTACTCACTGCTTCAGCTTGGACGCCTCGTTCTTAG

15 GENE1 (1596bp) ATGGATACTCTCTTTAGACTAGTCAGTCTCCAACAACAACAACAATCCGATAGTATCATT ACAAATCAATCTTCGTTAAGCAGAACTTCCACCACCACTACTGGCTCTCCACAAACTG CTTATCACTACAACTTTCCACAAAACGACGTCGTCGAAGAATGCTTCAACTTTTTCATG GATGAAGAAGACCTTTCCTCTTCTTCTTCTCACCACAACCATCACAACCACAACAATC CTAATACTTACTACTCTCCTTTCACTACTCCCACCCAATACCATCCCGCCACATCATCA ACCCCTTCCTCCACCGCCGCAGCCGCAGCTTTAGCCTCGCCTTACTCCTCCTCCGGC CACCATAATGACCCTTCCGCGTTCTCCATACCTCAAACTCCTCCGTCCTTCGACTTCT CAGCCAATGCCAAGTGGGCAGACTCGGTCCTTCTTGAAGCGGCACGTGCCTTCTCC GACAAAGACACTGCACGTGCGCAACAAATCCTATGGACGCTCAACGAGCTCTCTTCT CCGTACGGAGACACCGAGCAAAAACTGGCTTCTTACTTCCTCCAAGCTCTCTTCAAC CGCATGACCGGTTCAGGCGAACGATGCTACCGAACCATGGTAACAGCTGCAGCCAC AGAGAAGACTTGCTCCTTCGAGTCAACGCGAAAAACTGTACTAAAGTTCCAAGAAGTT AGCCCCTGGGCCACGTTTGGACACGTGGCGGCAAACGGAGCAATCTTGGAAGCAGT AGACGGAGAGGCAAAGATCCACATCGTTGACATAAGCTCCACGTTTTGCACTCAATG GCCGACTCTTCTAGAAGCTTTAGCCACAAGATCAGACGACACGCCTCACCTAAGGCT AACCACAGTTGTCGTGGCCAACAAGTTTGTCAACGATCAAACGGCGTCGCATCGGAT GATGAAAGAGATCGGAAACCGAATGGAGAAATTCGCTAGGCTTATGGGAGTTCCTTTC AAATTTAACATTATTCATCACGTTGGAGATTTATCTGAGTTTGATCTCAACGAACTCGAC GTTAAACCAGACGAAGTCTTGGCCATTAACTGCGTAGGCGCGATGCATGGGATCGCT TCACGTGGAAGCCCTAGAGACGCTGTGATATCGAGTTTCCGACGGTTAAGACCGAGG ATTGTGACGGTCGTAGAAGAAGAAGCTGATCTTGTCGGAGAAGAAGAAGGTGGCTTT GATGATGAGTTCTTGAGAGGGTTTGGAGAATGTTTACGATGGTTTAGGGTTTGCTTCG AGTCATGGGAAGAGAGTTTTCCAAGGACGAGCAACGAGAGGTTGATGCTAGAGCGT GCAGCGGGACGTGCGATCGTTGATCTTGTGGCTTGTGAGCCGTCGGATTCCACGGA GAGGCGAGAGACAGCGAGGAAGTGGTCGAGGAGGATGAGGAATAGTGGGTTTGGA GCGGTGGGGTATAGTGATGAGGTGGCGGATGATGTCAGAGCTTTGTTGAGGAGATAT AAAGAAGGTGTTTGGTCGATGGTACAGTGTCCTGATGCCGCCGGAATATTCCTTTGTT GGAGAGATCAGCCGGTGGTTTGGGCTAGTGCGTGGCGGCCAACGTAA

16 restriction enzyme site 찾는 법을 사용하여 size 예측 분자생물학실험 Materials & Method 294bp295bp 294bp295bp Gene1: 2076bp Vector: 3807bp Xba1- insert: cut at 1267bp - vector: cut at 346bp 1267 346 00 1267 346 Insert 의 방향이 역방향 Band size: 1318bp / 4565bp Insert 의 방향이 정방향 Band size: 860bp / 5023bp 2013-10-17

17 restriction enzyme site 찾는 법을 사용하여 size 예측 분자생물학실험 294bp295bp 294bp295bp Gene2: 1596bp Vector: 3807bp Pst1- insert: cut at 630bp - vector: cut at 310bp Apa1- vector: cut at 356bp 630 356 00 630 356 Insert 의 방향이 역방향 Band size: 46bp/ 645bp / 4711bp Insert 의 방향이 정방향 Band size: 46bp/ 981bp / 4375bp 2013-10-17 310 Materials & Method

18 18 분자생물학실험 Materials & Method Restriction Enzyme Double Address: http://pga.mgh.harvard.edu/web_apps/web_map/starthttp://pga.mgh.harvard.edu/web_apps/web_map/start

19 19 분자생물학실험 Materials & Method

20 20 분자생물학실험 Materials & Method

21 분자생물학실험 2013-10-10 Materials & Method Red: one cut Blue: more than one cut

22 분자생물학실험 Materials & Method

23 23 분자생물학실험 Materials & Method Restriction Enzyme Double

24 Miniprep solution Ⅰ, Ⅱ, Ⅲ 에 들어가는 시약의 역할, 왜 필요한지 ? 원리 Miniprep solution Ⅰ, Ⅱ, Ⅲ ( 아래 표 완성 ) Result & Discussion 분자생물학실험 Alkaline lysis solution Ⅰ ReagentFinal conc.Stock conc.Volume Glucose (MW=198.2)50mMpowder g Tris-Cl (pH8.0)25mM1M ml EDTA (pH8.0)10mM0.5M ml Total100ml

25 Result & Discussion 분자생물학실험 Alkaline lysis solution Ⅱ ReagentFinal conc.Stock conc.Volume NaOH0.2N10N ml SDS1%10% ml Total100ml Alkaline lysis solution Ⅲ ReagentFinal conc.Stock conc.Volume potassium acetate3M5M ml acetic acid99.9%11.5 ml D.W. Total100ml

26 1. 수업시간에 배웠던 restriction enzyme site 찾는 법을 사용하여 size 예 측 Result & Discussion 분자생물학실험 Insert Gene1 Control Enzyme1 개 Enzyme2 개 사용한 Enzyme 잘려지는 위치 Insert: Vector: Insert: Vector: 예상되는 band size

27 2. 새로운 restriction enzyme site 를 찾고 enzyme 이름 및 잘리는 위치, 결과로 확 인 될 band size 예측 ( 표 아래에 Enzyme 선택이유 (ex, 정방향, 역방향, band 확인 시 ) 쓰기 ) 3.Restriction enzyme 을 사용할 때 몇몇 enzyme 들은 BSA 를 필요로 한다. BSA 의 역할 조사 Result & Discussion 분자생물학실험 Insert Gene1Gene2 Enzyme1 개 Enzyme2 개 Enzyme1 개 Enzyme2 개 사용한 Enzyme 잘려지는 위치 Insert: Vector: Insert: Vector: Insert: Vector: Insert: Vector: 예상되는 band size

28 다음 실험 예비 레포트 분자생물학실험 - 이번 실험 Result&Discussion report -Sequencing 원리,