Team:KAIT Japan/Protocol

From 2012.igem.org

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__NOTOC__
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Editing now.
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='''Protocol'''=
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=='''Colony PCR'''==
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=Colony PCR=
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:'''Reagent'''
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Reagent
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:*TaKaRa Ex Taq(5units/μL) 0.5μL
:*TaKaRa Ex Taq(5units/μL) 0.5μL
:*10×Ex Taq buffer 10μL
:*10×Ex Taq buffer 10μL
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:*Template(E.coli DH5α)
:*Template(E.coli DH5α)
:*sterilized water(73.5μL)
:*sterilized water(73.5μL)
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Conditions of the thermal cycler
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:'''Conditions of the thermal cycler'''
#95°C(5min)
#95°C(5min)
#94°C(30sec)
#94°C(30sec)
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#*2-4:30cycle
#*2-4:30cycle
#*gradient:57-62°C(+0.1c)
#*gradient:57-62°C(+0.1c)
-
----
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=Ligation=
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=='''Ligation'''==
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Reagent
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:'''Reagent'''
:*sterilize water 2μL
:*sterilize water 2μL
:*PCR product 2μL
:*PCR product 2μL
:*vector DNA 1μL
:*vector DNA 1μL
:*Ligation Mighty Mix 5μL
:*Ligation Mighty Mix 5μL
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Method
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:'''Method'''
#Incubation(1h,16°C)
#Incubation(1h,16°C)
#Storage Overnight(-4°C)
#Storage Overnight(-4°C)
-
----
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-
=DNA extraction and purification of ''P.aeruginosa''=
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=='''DNA extraction and purification of ''P.aeruginosa'''''==
#Centrifuge culture medium(6,000rpm,5min,4°C)
#Centrifuge culture medium(6,000rpm,5min,4°C)
#Remove supernatant,Add saline[0.85%](1.5mL)
#Remove supernatant,Add saline[0.85%](1.5mL)
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#Pick up supernatant,remove new microtube
#Pick up supernatant,remove new microtube
#Add 3M-sodium acetate 40μL,chilled isopropanol 400μL
#Add 3M-sodium acetate 40μL,chilled isopropanol 400μL
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#Wind the DNA by a thin glass rod.
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#Wind the DNA by a thin glass rod
#Rinse chilled 70%-ethanol(500μL,about 30s)
#Rinse chilled 70%-ethanol(500μL,about 30s)
#Pick up DNA,air dry
#Pick up DNA,air dry
#Add TE buffer 200μL
#Add TE buffer 200μL
#*Melt DNA in buffer
#*Melt DNA in buffer
-
----
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-
=Transformation=
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=='''Transformation'''==
#Put competent cells on ice(10-15min)
#Put competent cells on ice(10-15min)
#Add Ligation reaction solution(10μL) and tapping
#Add Ligation reaction solution(10μL) and tapping
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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=
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=='''Confirmed of electrophoresis by PCR product and Ligation of the TA vector'''==
#Electrophoresis
#Electrophoresis
#*Gel concentration:1.2%,Migration time:30min
#*Gel concentration:1.2%,Migration time:30min
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#Heat insulation(16°C,30min)
#Heat insulation(16°C,30min)
#Storage(-20°C)
#Storage(-20°C)
-
----
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-
=The purified DNA=
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=='''The purified DNA'''==
#Electrophoresis
#Electrophoresis
#*Marker:dye 1μL,DNA molecule 2μL,TE buffer 3μL
#*Marker:dye 1μL,DNA molecule 2μL,TE buffer 3μL
#*Sample:dye 1μL,sample 5μL
#*Sample:dye 1μL,sample 5μL
#*Gel concentration:1.2%,Migration time:30min
#*Gel concentration:1.2%,Migration time:30min
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:::'''→Reflection:Band was less.'''
 
#Storage
#Storage
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----
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-
=PCR Product=
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=='''PCR Product'''==
#Electrophoresis
#Electrophoresis
#*The gel check and cut
#*The gel check and cut
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#*50cycle
#*50cycle
#Storage
#Storage
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----
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=Miniprep=
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=='''Miniprep'''==
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#Add culture medium 1mL in a microtube.
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#Add culture medium 1mL in a microtube
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#Centrifuge(1min,4°C,12,000rpm).
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#Centrifuge(1min,4°C,10,000rpm)
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#Remove the supernatant.
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#Remove the supernatant to new microtube
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#Repeat 1-3.
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#Repeat 1-3
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#Add SolI 100μL and Vortex.
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#Add SolI 100μL and Vortex
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#Centrifuge(1min,4°C,12,000rpm).
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#Centrifuge(1min,4°C,10,000rpm)
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#Add SolII 200μL and invert.
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#Add SolII 200μL and invert
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#ice-cold 3min.
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#ice-cold 3min
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#Add SolIII 150μL and invert.
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#Add SolIII 150μL and invert
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#ice-cold 5min.
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#ice-cold 5min
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#Centrifuge(5min,4°C,12,000rpm).
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#Centrifuge(5min,4°C,10,000rpm)
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#
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#Add the supernatant to new microtube
 +
#Add RNase 2μL
 +
#Incubation(20min,37°C)
 +
#Add phenol:chloroform 200μL
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#Tapping
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#Centrifuge(5min,4°C,10,000rpm)
 +
#Add the supernatant to new microtube
 +
#Add chloroform 200μL
 +
#Tapping
 +
#Centrifuge(1min,4°C,10,000rpm)
 +
#Add the supernatant(200μL) to new microtube
 +
#Add 3M-acetic acid 20μL
 +
#Add 100%Et 400μL and invert
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#Centrifuge(20min,4°C,10,000rpm)
 +
#Remove the supernatant to new microtube
 +
#Add 70%Et 400 μL
 +
#Tapping
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#Centrifuge(20min,4°C,10,000rpm)
 +
#Remove the supernatant to new microtube
 +
#Dry
 +
#Add TE buffer 50μL
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#Storage
|}
|}

Latest revision as of 06:00, 13 September 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

Kaitjapan team.png

Team

Protocol

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