Team:KAIT Japan/Protocol

From 2012.igem.org

(Difference between revisions)
Line 54: Line 54:
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Editing now.
 +
===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
 +
#95°C(5min)
 +
#94°C(30sec)
 +
#61°C(30sec)
 +
#71°C(40sec)
 +
#72°C(1min)
 +
#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
 +
#Incubation(1h,16°C)
 +
#Storage Overnight(-4°C)
 +
 +
===DNA extraction and purification of ''P.aeruginosa''===
 +
#Centrifuge culture medium(6,000rpm,5min,4°C)
 +
#Remove supernatant,Add saline[0.85%](1.5mL)
 +
#Centrifuge(6,000rpm,5min,4°C)
 +
#Add 5mMEDTA 1mL
 +
#Add 10%SDS 100μL
 +
#Add proteinase K 50methodL
 +
#Vortex
 +
#Incubation(30min,55°C)
 +
#Add phenol mixture(TE saturated phenol:chloroform:isoamyl alcohol=25:24:1)
 +
#Shake vigorously(1min)
 +
#*At this time,It became muddy white in color.
 +
#Centrifuge(16,000rpm,10min,4°C)
 +
#Pick up supernatant,remove new microtube
 +
#Repeat step 7-11
 +
#Add 3M-sodium acetate 40μL,chilled isopropanol 400μL
 +
#Vortex
 +
#Wind the DNA by a thin glass rod.
 +
#Rinse chilled 70%-ethanol(500μL)
 +
#Pick up DNA,air dry
 +
#Add TE buffer 500μL
 +
#Add RNase A 50μL
 +
#Incubation(20min,37°C)
 +
#Add proteinase K 50μL
 +
#Incubation(1h,37°C)
 +
#Add phenol mixture
 +
#Vortex(1min)
 +
#Centrifuge(16,000rpm,10min,4°C)
 +
#Pick up supernatant,remove new microtube
 +
#Add 3M-sodium acetate 40μL,chilled isopropanol 400μL
 +
#Wind the DNA by a thin glass rod.
 +
#Rinse chilled 70%-ethanol(500μL,about 30s)
 +
#Pick up DNA,air dry
 +
#Add TE buffer 200μL
 +
#*Melt DNA in buffer
 +
 +
===Transformation===
 +
#Put competent cells on ice(10-15min)
 +
#Add Ligation reaction solution(10μL) and tapping
 +
#On the ice(30min)[Transformation]
 +
#Add LB medium(0.7mL)
 +
#Incubate(60min,37°C)
 +
#Add X-gal(40μL) and ampicillin(10μL)[200μg/mL] on LB agar medium(IPTG)
 +
#Add one incubated(100μL)
 +
#Cultivation(overnight)
 +
 +
===Confirmed of electrophoresis by PCR product and Ligation of the TA vector===
 +
#Electrophoresis
 +
#*Gel concentration:1.2%,Migration time:30min
 +
#*Marker:Flash Gel 5μL
 +
#*Sample:dye 1μL,sample 5μL
 +
#Check and Colony PCR
 +
#Add to TA vector
 +
#*PCR product 2μL
 +
#*pMD20-Tvector 1μL
 +
#*D<sub>2</sub>W 2&mu;L
 +
#*Ligation Mighty Mix 5&mu;L
 +
#Heat insulation(16°C,30min)
 +
#Storage(-20°C)
 +
 +
===The purified DNA===
 +
#Electrophoresis
 +
#*Marker:dye 1&mu;L,DNA molecule 2&mu;L,TE buffer 3&mu;L
 +
#*Sample:dye 1&mu;L,sample 5&mu;L
 +
#*Gel concentration:1.2%,Migration time:30min
 +
:::'''→Reflection:Band was less.'''
 +
#Storage
 +
 +
===PCR Product===
 +
#Electrophoresis
 +
#*The gel check and cut
 +
#DNA purification
 +
#Confirmation of electrophoresis
 +
#PCR
 +
#*50cycle
 +
#Storage
 +
 +
===Miniprep===
 +
#Add culture medium 1mL in a microtube.
 +
#Centrifuge(1min,4°C,12,000rpm).
 +
#Remove the supernatant.
 +
#Repeat 1-3.
 +
#Add SolI 100&mu;L and Vortex.
 +
#Centrifuge(1min,4°C,12,000rpm).
 +
#Add SolII 200&mu;L and invert.
 +
#ice-cold 3min.
 +
#Add SolIII 150&mu;L and invert.
 +
#ice-cold 5min.
 +
#Centrifuge(5min,4°C,12,000rpm).
 +
#
|}
|}

Revision as of 08:14, 31 August 2012

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Home

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Project

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Parts

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Protocol

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Notebook

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Results

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Safety

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Human Practice

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Team

Editing now.

Contents

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).
Retrieved from "http://2012.igem.org/Team:KAIT_Japan/Protocol"