Team:KAIT Japan/Protocol

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<P>[[File:KAIT_Japan2012_logo.png|left|link=Team:KAIT_Japan]][[File:KAIT_Japan2012_Sub.png|right|link=http://www.kait.jp/index2.php]][[File:Kaitjapan_iGEM_official.logo.png‎|right|link=http://ung.igem.org/Main_Page]]
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[[Team:KAIT_Japan/Protocol|Protocol]]
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__NOTOC__
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='''Protocol'''=
 +
=='''Colony PCR'''==
 +
:'''Reagent'''
 +
:*TaKaRa Ex Taq(5units/&mu;L) 0.5&mu;L
 +
:*10×Ex Taq buffer 10&mu;L
 +
:*dNTP Mixture(2.5Meach) 8&mu;L
 +
:*Primer F(10&mu;M) 4&mu;L
 +
:*Primer R(10&mu;M) 4&mu;L
 +
:*Template(E.coli DH5α)
 +
:*sterilized water(73.5&mu;L)
 +
:'''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 2&mu;L
 +
:*PCR product 2&mu;L
 +
:*vector DNA 1&mu;L
 +
:*Ligation Mighty Mix 5&mu;L
 +
:'''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 100&mu;L
 +
#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 40&mu;L,chilled isopropanol 400&mu;L
 +
#Vortex
 +
#Wind the DNA by a thin glass rod.
 +
#Rinse chilled 70%-ethanol(500&mu;L)
 +
#Pick up DNA,air dry
 +
#Add TE buffer 500&mu;L
 +
#Add RNase A 50&mu;L
 +
#Incubation(20min,37°C)
 +
#Add proteinase K 50&mu;L
 +
#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 40&mu;L,chilled isopropanol 400&mu;L
 +
#Wind the DNA by a thin glass rod
 +
#Rinse chilled 70%-ethanol(500&mu;L,about 30s)
 +
#Pick up DNA,air dry
 +
#Add TE buffer 200&mu;L
 +
#*Melt DNA in buffer
 +
 +
=='''Transformation'''==
 +
#Put competent cells on ice(10-15min)
 +
#Add Ligation reaction solution(10&mu;L) and tapping
 +
#On the ice(30min)[Transformation]
 +
#Add LB medium(0.7mL)
 +
#Incubate(60min,37°C)
 +
#Add X-gal(40&mu;L) and ampicillin(10&mu;L)[200&mu;g/mL] on LB agar medium(IPTG)
 +
#Add one incubated(100&mu;L)
 +
#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 5&mu;L
 +
#*Sample:dye 1&mu;L,sample 5&mu;L
 +
#Check and Colony PCR
 +
#Add to TA vector
 +
#*PCR product 2&mu;L
 +
#*pMD20-Tvector 1&mu;L
 +
#*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
 +
#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,10,000rpm)
 +
#Remove the supernatant to new microtube
 +
#Repeat 1-3
 +
#Add SolI 100&mu;L and Vortex
 +
#Centrifuge(1min,4°C,10,000rpm)
 +
#Add SolII 200&mu;L and invert
 +
#ice-cold 3min
 +
#Add SolIII 150&mu;L and invert
 +
#ice-cold 5min
 +
#Centrifuge(5min,4°C,10,000rpm)
 +
#Add the supernatant to new microtube
 +
#Add RNase 2&mu;L
 +
#Incubation(20min,37°C)
 +
#Add phenol:chloroform 200&mu;L
 +
#Tapping
 +
#Centrifuge(5min,4°C,10,000rpm)
 +
#Add the supernatant to new microtube
 +
#Add chloroform 200&mu;L
 +
#Tapping
 +
#Centrifuge(1min,4°C,10,000rpm)
 +
#Add the supernatant(200&mu;L) to new microtube
 +
#Add 3M-acetic acid 20&mu;L
 +
#Add 100%Et 400&mu;L and invert
 +
#Centrifuge(20min,4°C,10,000rpm)
 +
#Remove the supernatant to new microtube
 +
#Add 70%Et 400 &mu;L
 +
#Tapping
 +
#Centrifuge(20min,4°C,10,000rpm)
 +
#Remove the supernatant to new microtube
 +
#Dry
 +
#Add TE buffer 50&mu;L
 +
#Storage
|}
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Latest revision as of 06:00, 13 September 2012

KAIT Japan2012 logo.png
KAIT Japan2012 Sub.png
Kaitjapan iGEM official.logo.png


Kaitjapan.home.png

Home

Kaitjapan project.png

Project

Kaitjapan parts.png

Parts

Kaitjapan protocol.png

Protocol

Kaitjapan notebook.png

Notebook

Kaitjapan results.png

Results

Kaitjapan safety.png

Safety

Kaitjapan human practice.png

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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