Team:KAIT Japan/Protocol

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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(5min)
2. 94°C(30sec)
3. 61°C(30sec)
4. 71°C(40sec)
5. 72°C(1min)
6. 4°C(Save)
   • 2-4:30cycle
   • gradient:57-62°C(+0.1c)

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)

DNA extraction and purification of P.aeruginosa

1. Centrifuge culture medium(6,000rpm,5min,4°C)
2. Remove supernatant,Add saline[0.85%](1.5mL)
3. Centrifuge(6,000rpm,5min,4°C)
4. Add 5mMEDTA 1mL
5. Add 10%SDS 100μL
6. Add proteinase K 50methodL
7. Vortex
8. Incubation(30min,55°C)
9. Add phenol mixture(TE saturated phenol:chloroform:isoamyl alcohol=25:24:1)
10. Shake vigorously(1min)
    • At this time,It became muddy white in color.
11. Centrifuge(16,000rpm,10min,4°C)
12. Pick up supernatant,remove new microtube
13. Repeat step 7-11
14. Add 3M-sodium acetate 40μL,chilled isopropanol 400μL
15. Vortex
16. Wind the DNA by a thin glass rod.
17. Rinse chilled 70%-ethanol(500μL)
18. Pick up DNA,air dry
19. Add TE buffer 500μL
20. Add RNase A 50μL
21. Incubation(20min,37°C)
22. Add proteinase K 50μL
23. Incubation(1h,37°C)
24. Add phenol mixture
25. Vortex(1min)
26. Centrifuge(16,000rpm,10min,4°C)
27. Pick up supernatant,remove new microtube
28. Add 3M-sodium acetate 40μL,chilled isopropanol 400μL
29. Wind the DNA by a thin glass rod.
30. Rinse chilled 70%-ethanol(500μL,about 30s)
31. Pick up DNA,air dry
32. Add TE buffer 200μL
    • Melt DNA in buffer

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)

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

→Reflection:Band was less.

1. Storage

PCR Product

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

Miniprep

1. Add culture medium 1mL in a microtube.
2. Centrifuge(1min,4°C,12,000rpm).
3. Remove the supernatant.
4. Repeat 1-3.
5. Add SolI 100μL and Vortex.
6. Centrifuge(1min,4°C,12,000rpm).
7. Add SolII 200μL and invert.
8. ice-cold 3min.
9. Add SolIII 150μL and invert.
10. ice-cold 5min.
11. Centrifuge(5min,4°C,12,000rpm).