1. 분자생물학실험
SUBJECT
Mini-prep,
Restriction Enzyme

2. 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.

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

4. 분자생물학실험
Minipreparation (Mini-prep)

5. 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.

6. [ 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 [

7. 분자생물학실험
Restriction Enzyme
insert
pTOP TA V2 R F

8. 분자생물학실험
294bp
295bp

9. pTOP TA V base pairs
agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattcgcccttaagggcgaattctgcagatatccatcacactggcggccgctcgagcatgcatctagagggcccaattcgccctatagtgaatcgtattacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctatacgtacggcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgccggggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaatgtcaggcatgagattatcaaaaaggatcttcacctagatccttttcacgtagaaagccagtccgcagaaacggtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgcagtgggcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagctctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagccaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggattcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgaatcagtgagcgaggaagcggaag

10. GENE1 (1596bp)
ATGGATACTCTCTTTAGACTAGTCAGTCTCCAACAACAACAACAATCCGATAGTATCATTACAAATCAATCTTCGTTAAGCAGAACTTCCACCACCACTACTGGCTCTCCACAAACTGCTTATCACTACAACTTTCCACAAAACGACGTCGTCGAAGAATGCTTCAACTTTTTCATGGATGAAGAAGACCTTTCCTCTTCTTCTTCTCACCACAACCATCACAACCACAACAATCCTAATACTTACTACTCTCCTTTCACTACTCCCACCCAATACCATCCCGCCACATCATCAACCCCTTCCTCCACCGCCGCAGCCGCAGCTTTAGCCTCGCCTTACTCCTCCTCCGGCCACCATAATGACCCTTCCGCGTTCTCCATACCTCAAACTCCTCCGTCCTTCGACTTCTCAGCCAATGCCAAGTGGGCAGACTCGGTCCTTCTTGAAGCGGCACGTGCCTTCTCCGACAAAGACACTGCACGTGCGCAACAAATCCTATGGACGCTCAACGAGCTCTCTTCTCCGTACGGAGACACCGAGCAAAAACTGGCTTCTTACTTCCTCCAAGCTCTCTTCAACCGCATGACCGGTTCAGGCGAACGATGCTACCGAACCATGGTAACAGCTGCAGCCACAGAGAAGACTTGCTCCTTCGAGTCAACGCGAAAAACTGTACTAAAGTTCCAAGAAGTTAGCCCCTGGGCCACGTTTGGACACGTGGCGGCAAACGGAGCAATCTTGGAAGCAGTAGACGGAGAGGCAAAGATCCACATCGTTGACATAAGCTCCACGTTTTGCACTCAATGGCCGACTCTTCTAGAAGCTTTAGCCACAAGATCAGACGACACGCCTCACCTAAGGCTAACCACAGTTGTCGTGGCCAACAAGTTTGTCAACGATCAAACGGCGTCGCATCGGATGATGAAAGAGATCGGAAACCGAATGGAGAAATTCGCTAGGCTTATGGGAGTTCCTTTCAAATTTAACATTATTCATCACGTTGGAGATTTATCTGAGTTTGATCTCAACGAACTCGACGTTAAACCAGACGAAGTCTTGGCCATTAACTGCGTAGGCGCGATGCATGGGATCGCTTCACGTGGAAGCCCTAGAGACGCTGTGATATCGAGTTTCCGACGGTTAAGACCGAGGATTGTGACGGTCGTAGAAGAAGAAGCTGATCTTGTCGGAGAAGAAGAAGGTGGCTTTGATGATGAGTTCTTGAGAGGGTTTGGAGAATGTTTACGATGGTTTAGGGTTTGCTTCGAGTCATGGGAAGAGAGTTTTCCAAGGACGAGCAACGAGAGGTTGATGCTAGAGCGTGCAGCGGGACGTGCGATCGTTGATCTTGTGGCTTGTGAGCCGTCGGATTCCACGGAGAGGCGAGAGACAGCGAGGAAGTGGTCGAGGAGGATGAGGAATAGTGGGTTTGGAGCGGTGGGGTATAGTGATGAGGTGGCGGATGATGTCAGAGCTTTGTTGAGGAGATATAAAGAAGGTGTTTGGTCGATGGTACAGTGTCCTGATGCCGCCGGAATATTCCTTTGTTGGAGAGATCAGCCGGTGGTTTGGGCTAGTGCGTGGCGGCCAACGTAA

11. Gene2 (2076bp) ATGGCGGAATCCGGCGATTTCAACGGTGGTCAACCTCCTCCTCATAGTCCTCTGAGAACAACTTCTTCCGGTAGTAGCAGCAGCAACAACCGTGGTCCTCCTCCTCCTCCTCCTCCTCCTTTAGTGATGGTGAGAAAAAGATTAGCTTCCGAGATGTCTTCTAACCCTGACTACAACAACTCCTCTCGTCCTCCTCGCCGTGTCTCTCACCTTCTTGACTCCAACTACAATACTGTCACACCACAACAACCACCGTCTCTTACGGCGGCGGCTACTGTATCTTCTCAACCAAACCCACCACTCTCTGTTTGTGGCTTCTCTGGTCTTCCCGTTTTTCCTTCAGACCGTGGTGGTCGGAATGTTATGATGTCCGTACAACCAATGGATCAAGACTCTTCATCTTCTTCTGCTTCACCTACTGTATGGGTTGACGCCATTATCAGAGACCTTATCCATTCCTCAACTTCAGTCTCTATTCCTCAACTTATCCAAAACGTTAGAGACATTATCTTCCCTTGTAACCCAAATCTCGGTGCTCTTCTTGAATACAGGCTCCGATCTCTCATGCTCCTTGATCCTTCCTCTTCCTCTGACCCTTCTCCTCAAACTTTCGAACCTCTCTATCAGATCTCCAACAATCCTTCTCCTCCACAACAGCAACAGCAGCACCAACAACAACAACAACAGCATAAGCCTCCTCCTCCTCCGATTCAGCAGCAAGAAAGAGAAAATTCTTCTACCGATGCACCACCGCAACCAGAGACAGTGACGGCCACTGTTCCCGCCGTCCAAACAAATACGGCGGAGGCTTTAAGAGAGAGGAAGGAAGAGATTAAGAGGCAGAAGCAAGACGAAGAAGGATTACACCTTCTCACATTGCTGCTACAGTGTGCTGAAGCTGTCTCTGCTGATAATCTCGAAGAAGCAAACAAGCTTCTTCTTGAGATCTCTCAGTTATCAACTCCTTACGGGACCTCAGCGCAGAGAGTAGCTGCTTACTTCTCGGAAGCTATGTCAGCGAGATTACTCAACTCGTGTCTCGGAATTTACGCGGCTTTGCCTTCACGGTGGATGCCTCAAACGCATAGCTTGAAAATGGTCTCTGCGTTTCAGGTCTTTAATGGGATAAGCCCTTTAGTGAAATTCTCACACTTTACAGCGAATCAGGCGATTCAAGAAGCATTTGAGAAAGAAGACAGTGTACACATCATTGACTTGGACATCATGCAGGGACTTCAATGGCCTGGTTTATTCCACATTCTTGCTTCTAGACCTGGAGGACCTCCACACGTGCGACTCACGGGACTTGGTACTTCCATGGAAGCTCTTCAGGCTACAGGGAAACGTCTTTCGGATTTCGCAGATAAGCTTGGCCTGCCTTTTGAGTTCTGCCCTTTAGCTGAGAAAGTTGGAAACTTGGACACTGAGAGACTCAATGTGAGGAAAAGGGAAGCTGTGGCTGTTCACTGGCTTCAACATTCTCTTTATGATGTCACTGGCTCTGATGCACACACTCTCTGGTTACTCCAAAGgtaaaataaacattaccttttaatcactctttatctataaattattttaagattatataggaaagatatgttctaaaaagtggcttttttggttaatgattggggaatgaacagATTAGCTCCTAAAGTTGTGACAGTAGTGGAACAAGATTTGAGCCACGCTGGTTCTTTCTTAGGAAGATTTGTAGAAGCAATACATTACTACTCTGCACTCTTTGACTCACTGGGAGCAAGCTACGGCGAAGAGAGTGAAGAGAGACATGTCGTGGAACAGCAGCTATTATCGAAAGAGATACGGAATGTATTAGCGGTTGGAGGACCATCGAGAAGCGGTGAAGTGAAGTTTGAGAGCTGGAGGGAGAAAATGCAACAATGTGGGTTTAAAGGTATATCTTTAGCTGGAAATGCAGCTACACAAGCGACTCTACTGTTGGGAATGTTTCCTTCGGATGGTTACACTTTGGTTGATGATAATGGTACACTTAAGCTTGGATGGAAAGATCTTTCGTTACTCACTGCTTCAGCTTGGACGCCTCGTTCTTAG

12. 분자생물학실험 Materials & Method Restriction Enzyme Double
Address:

13. 분자생물학실험
Materials & Method

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

15. 분자생물학실험
Materials & Method

16. 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

17. 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

18. 분자생물학실험
Materials & Method
Restriction Enzyme Double

19. 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

20. 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

21. 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

22. 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

23. 분자생물학실험
다음 실험 예비 레포트
Sequencing 원리,
Sequence blast 방법