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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)

Transformation

1. Put competent cells on ice(10-15min)
2. Add Ligation reaction solution(10μL) and tapping
3. On the ice(30min)[Transformation]
4. Add LB medium(0.7mL)
5. Incubate(60min,37°C)
6. Add X-gal(40μL) and ampicillin(10μL)[200μg/mL] on LB agar medium(IPTG)
7. Add one incubated(100μL)
8. Cultivation(overnight)

Ligation

Reagent

• sterilize water 2μL
• PCR product 2μL
• vector DNA 1μL
• Ligation Mighty Mix 5μL

Method

1. Incubation(1h,16°C)
2. Storage Overnight(-4°C)

Confirmed of electrophoresis by PCR product and Ligation of the TA vector

1. Electrophoresis
   • Gel concentration:1.2%,Migration time:30min
   • Marker:Flash Gel 5μL
   • Sample:dye 1μL,sample 5μL
2. Check and Colony PCR
3. Add to TA vector
   • PCR product 2μL
   • pMD20-Tvector 1μL
   • D₂W 2μL
   • Ligation Mighty Mix 5μL
4. Heat insulation(16°C,30min)
5. Storage(-20°C)

The purified DNA

1. Electrophoresis
   • Marker:dye 1μL,DNA molecule 2μL,TE buffer 3μL
   • Sample:dye 1μL,sample 5μL
   • Gel concentration:1.2%,Migration time:30min
2. Storage

PCR Product

1. Electrophoresis
   • The gel check and cut
2. DNA purification
3. Confirmation of electrophoresis
4. PCR
   • 50cycle
5. Storage

Infusion

1. Set up the In-Fusion cloning reaction:

• 5X In-Fusion HD Enzyme Premix 2 μl
• Linearized Vector x μl*
• Cloning Enhancer-Treated PCR Fragment x μl**
• dH2O (as needed) x μl
• Total Volume 10 μl

*Use 50–200 ng of linearized vector.

**Use 1–2 μl of Cloning Enhancer-Treated fragments, regardless of their length. The total volume of Cloning Enhancer-Treated PCR fragments should be up to 4 μl per 10 μl reaction. If you obtain a low product yield from your PCR reac¬tion, we recommend purification of PCR fragments instead of Cloning Enhancer-Treatment.

1. Adjust the total reaction volume to 10 μl using deionized H2. 2O and mix the reaction.
2. Incubate the reaction for 15 min at 50°C, then place on ice.
3. Continue to the Transformation Procedure (Section VIII). If you cannot transform cells immediately, store the cloning reactions at –20°C until you are ready.