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Team:KAIT Japan/Protocol
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| + | __NOTOC__ | ||
| + | ='''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''' | ||
| + | #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 2μL | ||
| + | :*PCR product 2μL | ||
| + | :*vector DNA 1μL | ||
| + | :*Ligation Mighty Mix 5μL | ||
| + | :'''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 100μL | ||
| + | #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 40μL,chilled isopropanol 400μL | ||
| + | #Vortex | ||
| + | #Wind the DNA by a thin glass rod. | ||
| + | #Rinse chilled 70%-ethanol(500μL) | ||
| + | #Pick up DNA,air dry | ||
| + | #Add TE buffer 500μL | ||
| + | #Add RNase A 50μL | ||
| + | #Incubation(20min,37°C) | ||
| + | #Add proteinase K 50μL | ||
| + | #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 40μL,chilled isopropanol 400μL | ||
| + | #Wind the DNA by a thin glass rod | ||
| + | #Rinse chilled 70%-ethanol(500μL,about 30s) | ||
| + | #Pick up DNA,air dry | ||
| + | #Add TE buffer 200μL | ||
| + | #*Melt DNA in buffer | ||
| + | |||
| + | =='''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) | ||
| + | |||
| + | =='''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 2μL | ||
| + | #*Ligation Mighty Mix 5μL | ||
| + | #Heat insulation(16°C,30min) | ||
| + | #Storage(-20°C) | ||
| + | |||
| + | =='''The purified DNA'''== | ||
| + | #Electrophoresis | ||
| + | #*Marker:dye 1μL,DNA molecule 2μL,TE buffer 3μL | ||
| + | #*Sample:dye 1μL,sample 5μL | ||
| + | #*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 100μL and Vortex | ||
| + | #Centrifuge(1min,4°C,10,000rpm) | ||
| + | #Add SolII 200μL and invert | ||
| + | #ice-cold 3min | ||
| + | #Add SolIII 150μL and invert | ||
| + | #ice-cold 5min | ||
| + | #Centrifuge(5min,4°C,10,000rpm) | ||
| + | #Add the supernatant to new microtube | ||
| + | #Add RNase 2μL | ||
| + | #Incubation(20min,37°C) | ||
| + | #Add phenol:chloroform 200μL | ||
| + | #Tapping | ||
| + | #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 | ||
| + | #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 | ||
|} | |} | ||
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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