Team:KAIT Japan/Protocol

From 2012.igem.org

(Difference between revisions)
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#*2-4:30cycle
#*2-4:30cycle
#*gradient:57-62°C(+0.1c)
#*gradient:57-62°C(+0.1c)
-
 
+
----
===Ligation===
===Ligation===
Reagent
Reagent
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#Incubation(1h,16°C)
#Incubation(1h,16°C)
#Storage Overnight(-4°C)
#Storage Overnight(-4°C)
-
 
+
----
===DNA extraction and purification of ''P.aeruginosa''===
===DNA extraction and purification of ''P.aeruginosa''===
#Centrifuge culture medium(6,000rpm,5min,4°C)
#Centrifuge culture medium(6,000rpm,5min,4°C)
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#Add TE buffer 200μL
#Add TE buffer 200μL
#*Melt DNA in buffer
#*Melt DNA in buffer
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+
----
===Transformation===
===Transformation===
#Put competent cells on ice(10-15min)
#Put competent cells on ice(10-15min)
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#Add one incubated(100μL)
#Add one incubated(100μL)
#Cultivation(overnight)
#Cultivation(overnight)
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+
----
===Confirmed of electrophoresis by PCR product and Ligation of the TA vector===
===Confirmed of electrophoresis by PCR product and Ligation of the TA vector===
#Electrophoresis
#Electrophoresis
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#Heat insulation(16°C,30min)
#Heat insulation(16°C,30min)
#Storage(-20°C)
#Storage(-20°C)
-
 
+
----
===The purified DNA===
===The purified DNA===
#Electrophoresis
#Electrophoresis
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:::'''→Reflection:Band was less.'''
:::'''→Reflection:Band was less.'''
#Storage
#Storage
-
 
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----
===PCR Product===
===PCR Product===
#Electrophoresis
#Electrophoresis
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#*50cycle
#*50cycle
#Storage
#Storage
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+
----
===Miniprep===
===Miniprep===
#Add culture medium 1mL in a microtube.
#Add culture medium 1mL in a microtube.

Revision as of 08:16, 31 August 2012

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Home

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Project

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Parts

Kaitjapan protocol.png

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"