Team:KAIT Japan/Protocol

From 2013.igem.org

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:[[File:KAIT Japan human practice.png|link=Team:KAIT_Japan/Human Practice]] [[Team:KAIT_Japan/Human Practice|Human Practice]]
:[[File:KAIT Japan human practice.png|link=Team:KAIT_Japan/Human Practice]] [[Team:KAIT_Japan/Human Practice|Human Practice]]
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='''Protocol'''=
='''Protocol'''=
 +
__NOTOC__
 +
=='''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 100μL and Vortex
 +
#Add SolII 200μL and invert
 +
#ice-cold 3min
 +
#Add SolIII 150μL and invert
 +
#ice-cold 3min
 +
#Centrifuge(10min,4°C,10,000rpm)
 +
#Add the supernatant to new microtube
 +
#Add RNase 0.8μL
 +
#Incubation(1h,37°C)
 +
#Add phenol:chloroform 200μL
 +
#Vortex
 +
#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(150μL) to new microtube
 +
#Add 3M-acetic acid 15μL
 +
#Add 100%Et 400μL and invert
 +
#Centrifuge(20min,4°C,10,000rpm)
 +
#Remove the supernatant to new microtube
 +
#Add 70%Et 400 μL
 +
#Tapping
 +
#Centrifuge(20min,4°C,10,000rpm)
 +
#Remove the supernatant to new microtube
 +
#Dry
 +
#Add TE buffer 50μL
 +
#Storage
 +
=='''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(2min)
 +
#95°C(30sec)
 +
#54°C(30sec)
 +
#72°C(40sec)
 +
#72°C(5min)
 +
#4°C(Save)
 +
#*2-4:40cycle
 +
#*gradient:57-62°C(+0.1c)
 +
=='''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)
 +
=='''Ligation'''==
 +
::Reagent
 +
:*sterilize water 2μL
 +
:*PCR product 2μL
 +
:*vector DNA 1μL
 +
:*Ligation Mighty Mix 5μL
 +
::Method
 +
#Incubation(1h,16°C)
 +
#Storage Overnight(-4°C)
 +
=='''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
 +
#Storage
 +
=='''PCR Product'''==
 +
#Electrophoresis
 +
#*The gel check and cut
 +
#DNA purification
 +
#Confirmation of electrophoresis
 +
#PCR
 +
#*50cycle
 +
#Storage
-
 
+
=='''Infusion'''==
 +
#Set up the In-Fusion cloning reaction:
 +
:::*5X In-Fusion HD Enzyme Premix 2 μl
 +
:::*Linearized Vector x μl*
 +
:::*Cloning Enhancer-Treated PCR Fragment x μl**
 +
:::*dH2O (as needed) x μl
 +
:::*Total Volume 10 μl
 +
:: *Use 50–200 ng of linearized vector.
 +
:: **Use 1–2 μl of Cloning Enhancer-Treated fragments, regardless of their length. The total volume of Cloning Enhancer-Treated PCR fragments should be up to 4 μl per 10 μl reaction. If you obtain a low product yield from your PCR reac¬tion, we recommend purification of PCR fragments instead of Cloning Enhancer-Treatment.
 +
#Adjust the total reaction volume to 10 μl using deionized H2. 2O and mix the reaction.
 +
#Incubate the reaction for 15 min at 50°C, then place on ice.
 +
#Continue to the Transformation Procedure (Section VIII). If you cannot transform cells immediately, store the  cloning reactions at –20°C until you are ready.
|}
|}

Latest revision as of 06:08, 9 September 2013

KAIT Japan 2013RogoTop.png

KAIT Japan2013 Kanako.png KAIT Japan 2013 iGEMRogo.png

KAIT Japan home.png Home
KAIT Japan team.png Team
KAIT Japan project.png Project
KAIT Japan parts.png Parts

KAIT_Japan_protocol.png Protocol

KAIT Japan notebook.png Notebook
KAIT Japan results.png Results
KAIT Japan safety.png Safety
KAIT Japan human practice.png Human Practice

Protocol

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. Add SolII 200μL and invert
  7. ice-cold 3min
  8. Add SolIII 150μL and invert
  9. ice-cold 3min
  10. Centrifuge(10min,4°C,10,000rpm)
  11. Add the supernatant to new microtube
  12. Add RNase 0.8μL
  13. Incubation(1h,37°C)
  14. Add phenol:chloroform 200μL
  15. Vortex
  16. Centrifuge(5min,4°C,10,000rpm)
  17. Add the supernatant to new microtube
  18. Add chloroform 200μL
  19. Tapping
  20. Centrifuge(1min,4°C,10,000rpm)
  21. Add the supernatant(150μL) to new microtube
  22. Add 3M-acetic acid 15μL
  23. Add 100%Et 400μL and invert
  24. Centrifuge(20min,4°C,10,000rpm)
  25. Remove the supernatant to new microtube
  26. Add 70%Et 400 μL
  27. Tapping
  28. Centrifuge(20min,4°C,10,000rpm)
  29. Remove the supernatant to new microtube
  30. Dry
  31. Add TE buffer 50μL
  32. Storage

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(2min)
  2. 95°C(30sec)
  3. 54°C(30sec)
  4. 72°C(40sec)
  5. 72°C(5min)
  6. 4°C(Save)
    • 2-4:40cycle
    • gradient:57-62°C(+0.1c)

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)

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)

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

Infusion

  1. Set up the In-Fusion cloning reaction:
  • 5X In-Fusion HD Enzyme Premix 2 μl
  • Linearized Vector x μl*
  • Cloning Enhancer-Treated PCR Fragment x μl**
  • dH2O (as needed) x μl
  • Total Volume 10 μl
*Use 50–200 ng of linearized vector.
**Use 1–2 μl of Cloning Enhancer-Treated fragments, regardless of their length. The total volume of Cloning Enhancer-Treated PCR fragments should be up to 4 μl per 10 μl reaction. If you obtain a low product yield from your PCR reac¬tion, we recommend purification of PCR fragments instead of Cloning Enhancer-Treatment.
  1. Adjust the total reaction volume to 10 μl using deionized H2. 2O and mix the reaction.
  2. Incubate the reaction for 15 min at 50°C, then place on ice.
  3. Continue to the Transformation Procedure (Section VIII). If you cannot transform cells immediately, store the cloning reactions at –20°C until you are ready.
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