Team:KAIT Japan/Protocol
From 2012.igem.org
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- | + | __NOTOC__ | |
- | =Colony PCR= | + | ='''Protocol'''= |
- | Reagent | + | =='''Colony PCR'''== |
+ | :'''Reagent''' | ||
:*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) | ||
- | Conditions of the thermal cycler | + | :'''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) | ||
- | + | ||
- | =Ligation= | + | =='''Ligation'''== |
- | Reagent | + | :'''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 | ||
- | Method | + | :'''Method''' |
#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) | ||
#Remove supernatant,Add saline[0.85%](1.5mL) | #Remove supernatant,Add saline[0.85%](1.5mL) | ||
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#Add TE buffer 200μL | #Add TE buffer 200μL | ||
#*Melt DNA in buffer | #*Melt DNA in buffer | ||
- | |||
- | =Transformation= | + | =='''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) | ||
- | + | ||
- | =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 | ||
#*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) | ||
- | + | ||
- | =The purified DNA= | + | =='''The purified DNA'''== |
#Electrophoresis | #Electrophoresis | ||
#*Marker:dye 1μL,DNA molecule 2μL,TE buffer 3μL | #*Marker:dye 1μL,DNA molecule 2μL,TE buffer 3μL | ||
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#*Gel concentration:1.2%,Migration time:30min | #*Gel concentration:1.2%,Migration time:30min | ||
#Storage | #Storage | ||
- | + | ||
- | =PCR Product= | + | =='''PCR Product'''== |
#Electrophoresis | #Electrophoresis | ||
#*The gel check and cut | #*The gel check and cut | ||
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#*50cycle | #*50cycle | ||
#Storage | #Storage | ||
- | + | ||
- | =Miniprep= | + | =='''Miniprep'''== |
#Add culture medium 1mL in a microtube | #Add culture medium 1mL in a microtube | ||
#Centrifuge(1min,4°C,10,000rpm) | #Centrifuge(1min,4°C,10,000rpm) |
Latest revision as of 06:00, 13 September 2012
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ProtocolColony PCR
Ligation
DNA extraction and purification of P.aeruginosa
Transformation
Confirmed of electrophoresis by PCR product and Ligation of the TA vector
The purified DNA
PCR Product
Miniprep
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