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Safety

Human Practice

Protocol

Miniprep

1. Add culture medium 1mL in a microtube
2. Centrifuge(1min,4°C,10,000rpm)
3. Remove the supernatant to new microtube
4. Repeat 1-3
5. Add SolI 100μL and Vortex
6. Add SolII 200μL and invert
7. ice-cold 3min
8. Add SolIII 150μL and invert
9. ice-cold 3min
10. Centrifuge(10min,4°C,10,000rpm)
11. Add the supernatant to new microtube
12. Add RNase 0.8μL
13. Incubation(1h,37°C)
14. Add phenol:chloroform 200μL
15. Vortex
16. Centrifuge(5min,4°C,10,000rpm)
17. Add the supernatant to new microtube
18. Add chloroform 200μL
19. Tapping
20. Centrifuge(1min,4°C,10,000rpm)
21. Add the supernatant(150μL) to new microtube
22. Add 3M-acetic acid 15μL
23. Add 100%Et 400μL and invert
24. Centrifuge(20min,4°C,10,000rpm)
25. Remove the supernatant to new microtube
26. Add 70%Et 400 μL
27. Tapping
28. Centrifuge(20min,4°C,10,000rpm)
29. Remove the supernatant to new microtube
30. Dry
31. Add TE buffer 50μL
32. Storage

Colony PCR

Reagent

• TaKaRa Ex Taq(5units/μL) 0.5μL
• 10×Ex Taq buffer 10μL
• dNTP Mixture(2.5Meach) 8μL
• Primer F(10μM) 4μL
• Primer R(10μM) 4μL
• Template(E.coli DH5α)
• sterilized water(73.5μL)

Conditions of the thermal cycler

1. 95°C(2min)
2. 95°C(30sec)
3. 54°C(30sec)
4. 72°C(40sec)
5. 72°C(5min)
6. 4°C(Save)
   • 2-4:40cycle
   • gradient:57-62°C(+0.1c)