분자생물학실험 SUBJECT Mini-prep, Restriction Enzyme 2014-10-08

분자생물학실험 TA cloning Materials & Method A schematic of pTOP V2(3807 bp). A molecule of topoisomerase(TOPO) is covalently coupled to each of the two 3. one-nt protruding ends (nt positions 294 and 295)of pTOP TA. Therefore, pTOP TA V2 is ready to form a covalent bond when mixed with PCR products with one-nt 3. A overhang. The addition of one A to the duplex DNA ends is intrinsic to Taq DNA polymerase.

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

분자생물학실험 Minipreparation (Mini-prep)

분자생물학실험 Materials & Method Minipreparation (Mini-prep) Alkaline lysis solution Ⅰ autoclave for 15min and store at 4℃ Reagent conc. Glucose (MW=198.2) 50mM Tris-Cl (pH8.0) 25mM EDTA (pH8.0) 10mM Alkaline lysis solution Ⅱ 0.2N NaOH (freshly diluted from a 10N stock) Prepare Sol2 fresh and use at room temperature. Reagent conc. NaOH 0.2N SDS 1% Alkaline lysis solution Ⅲ store at 4℃ and transfer it to an ice just before use. Reagent conc. potassium acetate 3M acetic acid D.W.

[ 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 SolⅠ by vortexing. 5. Transfer the 100ul 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℃ 10. Transfer the 350ul supernatant into new e-tube and precipitate by 2 volumes of ethanol. 11. Centrifuge at 13,000rpm for 5min at 4℃ 12. Remove the supernatant and add 1mL 70% Ethanol. 13. Centrifuge at 13,000rpm for 2min at 4℃ 14. Dry the DNA pellet 15. Dissolve DNA in 30ul D.W. with 20ug/ml RNase A 16. 37℃ incubation for 30min [

분자생물학실험 Restriction Enzyme insert pTOP TA V2 R F

분자생물학실험 294bp 295bp

pTOP TA V2 3807 base pairs agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattcgcccttaagggcgaattctgcagatatccatcacactggcggccgctcgagcatgcatctagagggcccaattcgccctatagtgaatcgtattacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctatacgtacggcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgccggggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaatgtcaggcatgagattatcaaaaaggatcttcacctagatccttttcacgtagaaagccagtccgcagaaacggtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgcagtgggcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagctctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagccaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggattcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgaatcagtgagcgaggaagcggaag

GENE1 (1596bp) ATGGATACTCTCTTTAGACTAGTCAGTCTCCAACAACAACAACAATCCGATAGTATCATTACAAATCAATCTTCGTTAAGCAGAACTTCCACCACCACTACTGGCTCTCCACAAACTGCTTATCACTACAACTTTCCACAAAACGACGTCGTCGAAGAATGCTTCAACTTTTTCATGGATGAAGAAGACCTTTCCTCTTCTTCTTCTCACCACAACCATCACAACCACAACAATCCTAATACTTACTACTCTCCTTTCACTACTCCCACCCAATACCATCCCGCCACATCATCAACCCCTTCCTCCACCGCCGCAGCCGCAGCTTTAGCCTCGCCTTACTCCTCCTCCGGCCACCATAATGACCCTTCCGCGTTCTCCATACCTCAAACTCCTCCGTCCTTCGACTTCTCAGCCAATGCCAAGTGGGCAGACTCGGTCCTTCTTGAAGCGGCACGTGCCTTCTCCGACAAAGACACTGCACGTGCGCAACAAATCCTATGGACGCTCAACGAGCTCTCTTCTCCGTACGGAGACACCGAGCAAAAACTGGCTTCTTACTTCCTCCAAGCTCTCTTCAACCGCATGACCGGTTCAGGCGAACGATGCTACCGAACCATGGTAACAGCTGCAGCCACAGAGAAGACTTGCTCCTTCGAGTCAACGCGAAAAACTGTACTAAAGTTCCAAGAAGTTAGCCCCTGGGCCACGTTTGGACACGTGGCGGCAAACGGAGCAATCTTGGAAGCAGTAGACGGAGAGGCAAAGATCCACATCGTTGACATAAGCTCCACGTTTTGCACTCAATGGCCGACTCTTCTAGAAGCTTTAGCCACAAGATCAGACGACACGCCTCACCTAAGGCTAACCACAGTTGTCGTGGCCAACAAGTTTGTCAACGATCAAACGGCGTCGCATCGGATGATGAAAGAGATCGGAAACCGAATGGAGAAATTCGCTAGGCTTATGGGAGTTCCTTTCAAATTTAACATTATTCATCACGTTGGAGATTTATCTGAGTTTGATCTCAACGAACTCGACGTTAAACCAGACGAAGTCTTGGCCATTAACTGCGTAGGCGCGATGCATGGGATCGCTTCACGTGGAAGCCCTAGAGACGCTGTGATATCGAGTTTCCGACGGTTAAGACCGAGGATTGTGACGGTCGTAGAAGAAGAAGCTGATCTTGTCGGAGAAGAAGAAGGTGGCTTTGATGATGAGTTCTTGAGAGGGTTTGGAGAATGTTTACGATGGTTTAGGGTTTGCTTCGAGTCATGGGAAGAGAGTTTTCCAAGGACGAGCAACGAGAGGTTGATGCTAGAGCGTGCAGCGGGACGTGCGATCGTTGATCTTGTGGCTTGTGAGCCGTCGGATTCCACGGAGAGGCGAGAGACAGCGAGGAAGTGGTCGAGGAGGATGAGGAATAGTGGGTTTGGAGCGGTGGGGTATAGTGATGAGGTGGCGGATGATGTCAGAGCTTTGTTGAGGAGATATAAAGAAGGTGTTTGGTCGATGGTACAGTGTCCTGATGCCGCCGGAATATTCCTTTGTTGGAGAGATCAGCCGGTGGTTTGGGCTAGTGCGTGGCGGCCAACGTAA

Gene2 (2076bp) ATGGCGGAATCCGGCGATTTCAACGGTGGTCAACCTCCTCCTCATAGTCCTCTGAGAACAACTTCTTCCGGTAGTAGCAGCAGCAACAACCGTGGTCCTCCTCCTCCTCCTCCTCCTCCTTTAGTGATGGTGAGAAAAAGATTAGCTTCCGAGATGTCTTCTAACCCTGACTACAACAACTCCTCTCGTCCTCCTCGCCGTGTCTCTCACCTTCTTGACTCCAACTACAATACTGTCACACCACAACAACCACCGTCTCTTACGGCGGCGGCTACTGTATCTTCTCAACCAAACCCACCACTCTCTGTTTGTGGCTTCTCTGGTCTTCCCGTTTTTCCTTCAGACCGTGGTGGTCGGAATGTTATGATGTCCGTACAACCAATGGATCAAGACTCTTCATCTTCTTCTGCTTCACCTACTGTATGGGTTGACGCCATTATCAGAGACCTTATCCATTCCTCAACTTCAGTCTCTATTCCTCAACTTATCCAAAACGTTAGAGACATTATCTTCCCTTGTAACCCAAATCTCGGTGCTCTTCTTGAATACAGGCTCCGATCTCTCATGCTCCTTGATCCTTCCTCTTCCTCTGACCCTTCTCCTCAAACTTTCGAACCTCTCTATCAGATCTCCAACAATCCTTCTCCTCCACAACAGCAACAGCAGCACCAACAACAACAACAACAGCATAAGCCTCCTCCTCCTCCGATTCAGCAGCAAGAAAGAGAAAATTCTTCTACCGATGCACCACCGCAACCAGAGACAGTGACGGCCACTGTTCCCGCCGTCCAAACAAATACGGCGGAGGCTTTAAGAGAGAGGAAGGAAGAGATTAAGAGGCAGAAGCAAGACGAAGAAGGATTACACCTTCTCACATTGCTGCTACAGTGTGCTGAAGCTGTCTCTGCTGATAATCTCGAAGAAGCAAACAAGCTTCTTCTTGAGATCTCTCAGTTATCAACTCCTTACGGGACCTCAGCGCAGAGAGTAGCTGCTTACTTCTCGGAAGCTATGTCAGCGAGATTACTCAACTCGTGTCTCGGAATTTACGCGGCTTTGCCTTCACGGTGGATGCCTCAAACGCATAGCTTGAAAATGGTCTCTGCGTTTCAGGTCTTTAATGGGATAAGCCCTTTAGTGAAATTCTCACACTTTACAGCGAATCAGGCGATTCAAGAAGCATTTGAGAAAGAAGACAGTGTACACATCATTGACTTGGACATCATGCAGGGACTTCAATGGCCTGGTTTATTCCACATTCTTGCTTCTAGACCTGGAGGACCTCCACACGTGCGACTCACGGGACTTGGTACTTCCATGGAAGCTCTTCAGGCTACAGGGAAACGTCTTTCGGATTTCGCAGATAAGCTTGGCCTGCCTTTTGAGTTCTGCCCTTTAGCTGAGAAAGTTGGAAACTTGGACACTGAGAGACTCAATGTGAGGAAAAGGGAAGCTGTGGCTGTTCACTGGCTTCAACATTCTCTTTATGATGTCACTGGCTCTGATGCACACACTCTCTGGTTACTCCAAAGgtaaaataaacattaccttttaatcactctttatctataaattattttaagattatataggaaagatatgttctaaaaagtggcttttttggttaatgattggggaatgaacagATTAGCTCCTAAAGTTGTGACAGTAGTGGAACAAGATTTGAGCCACGCTGGTTCTTTCTTAGGAAGATTTGTAGAAGCAATACATTACTACTCTGCACTCTTTGACTCACTGGGAGCAAGCTACGGCGAAGAGAGTGAAGAGAGACATGTCGTGGAACAGCAGCTATTATCGAAAGAGATACGGAATGTATTAGCGGTTGGAGGACCATCGAGAAGCGGTGAAGTGAAGTTTGAGAGCTGGAGGGAGAAAATGCAACAATGTGGGTTTAAAGGTATATCTTTAGCTGGAAATGCAGCTACACAAGCGACTCTACTGTTGGGAATGTTTCCTTCGGATGGTTACACTTTGGTTGATGATAATGGTACACTTAAGCTTGGATGGAAAGATCTTTCGTTACTCACTGCTTCAGCTTGGACGCCTCGTTCTTAG

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

분자생물학실험 Materials & Method

분자생물학실험 Materials & Method Red: one cut Blue: more than one cut

분자생물학실험 Materials & Method

분자생물학실험 restriction enzyme site 찾는 법을 사용하여 size예측 Example 294bp 295bp 295bp 294bp 295bp 1267 1267 346 346 Gene2: 2076bp Vector: 3807bp Xba1- insert: cut at 1267bp - vector: cut at 346bp Insert의 방향이 정방향 Band size: 860bp / 5023bp Insert의 방향이 역방향 Band size: 1318bp / 4565bp

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

분자생물학실험 Materials & Method Restriction Enzyme Double

분자생물학실험 Materials & Method Restriction enzyme (mixture) Gene1 Gene2 10x Buffer 1㎕ Enzyme(Sac I) 0.2㎕ 10x BSA(1/10) Template 5㎕ DW Up to 10㎕ Total 10㎕ 10x Buffer 1㎕ Enzyme (Nco I) 0.2㎕ Enzyme (Sac I) 10x BSA(1/10) Template 5㎕ DW Up to 10㎕ Total 10㎕ PCR tube 에 위의 mixture 넣고 37℃ chamber에 1~2hr 보관

분자생물학실험 Result & Discussion Miniprep solution Ⅰ, Ⅱ, Ⅲ 에 들어가는 시약의 역할, 왜 필요한지? 원리 Miniprep solution Ⅰ, Ⅱ, Ⅲ ( 아래 표 완성 ) Alkaline lysis solution Ⅰ Reagent Final conc. Stock conc. Volume Glucose (MW=198.2) 50mM powder g Tris-Cl (pH8.0) 25mM 1M ml EDTA (pH8.0) 10mM 0.5M D.W. ___ml Total 100ml

분자생물학실험 Result & Discussion Alkaline lysis solution Ⅱ Reagent Final conc. Stock conc. Volume NaOH 0.2N 10N ml SDS 1% 10% D.W. ___ml Total 100ml Alkaline lysis solution Ⅲ Reagent Final conc. Stock conc. Volume potassium acetate 3M 5M ml acetic acid 99.9% 11.5 ml D.W. ___ml Total 100ml

분자생물학실험 Result & Discussion 수업시간에 배웠던 restriction enzyme site 찾는 법을 사용하여 size예측 새로운 restriction enzyme site를 찾고 (각각 1개씩) enzyme 이름 및 size예측 Restriction enzyme을 사용할 때 몇몇 enzyme들은 BSA를 필요로 한다. BSA의 역할 조사

분자생물학실험 다음 실험 예비 레포트 Sequencing 원리, Sequence blast 방법