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Team:KAIT Japan/Notebook
From 2012.igem.org
(Difference between revisions)
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===Colony PCR=== | ===Colony PCR=== | ||
Reagent | Reagent | ||
| - | :*TaKaRa Ex Taq(5units/ | + | :*TaKaRa Ex Taq(5units/μL) 0.5μL |
| - | :*10×Ex Taq buffer | + | :*10×Ex Taq buffer 10μL |
| - | :*dNTP Mixture(2.5Meach) | + | :*dNTP Mixture(2.5Meach) 8μL |
| - | :*Primer F( | + | :*Primer F(10μM) 4μL |
| - | :*Primer R( | + | :*Primer R(10μM) 4μL |
:*Template(E.coli DH5α) | :*Template(E.coli DH5α) | ||
::'''→Reflection:We Took E.coli too many.You should take less E.coli.'''<br> | ::'''→Reflection:We Took E.coli too many.You should take less E.coli.'''<br> | ||
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===The purified DNA=== | ===The purified DNA=== | ||
#Electrophoresis | #Electrophoresis | ||
| - | #*Marker:dye | + | #*Marker:dye 1μL,DNA molecule 2μL,TE buffer 3μL |
| - | #*Sample:dye | + | #*Sample:dye 1μL,sample 5μL |
#*Gel concentration:1.2%,Migration time:30min | #*Gel concentration:1.2%,Migration time:30min | ||
:::'''→Reflection:Band was less.''' | :::'''→Reflection:Band was less.''' | ||
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#Electrophoresis | #Electrophoresis | ||
#*Gel concentration:1.2%,Migration time:30min | #*Gel concentration:1.2%,Migration time:30min | ||
| - | #*Marker:Flash Gel | + | #*Marker:Flash Gel 5μL |
| - | #*Sample:dye | + | #*Sample:dye 1μL,sample 5μL |
#Check and Colony PCR | #Check and Colony PCR | ||
#Add to TA vector | #Add to TA vector | ||
| - | #*PCR product | + | #*PCR product 2μL |
| - | #*pMD20-Tvector | + | #*pMD20-Tvector 1μL |
| - | #*D<sub>2</sub>W | + | #*D<sub>2</sub>W 2μL |
| - | #*Ligation Mighty Mix | + | #*Ligation Mighty Mix 5μL |
#Heat insulation(16°C,30min) | #Heat insulation(16°C,30min) | ||
#Storage(-20°C) | #Storage(-20°C) | ||
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===Transformation=== | ===Transformation=== | ||
#Put competent cells on ice(10-15min) | #Put competent cells on ice(10-15min) | ||
| - | #Add Ligation reaction solution( | + | #Add Ligation reaction solution(10μL) and tapping |
#On the ice(30min)[Transformation] | #On the ice(30min)[Transformation] | ||
#Add LB medium(0.7mL) | #Add LB medium(0.7mL) | ||
#Incubate(60min,37°C) | #Incubate(60min,37°C) | ||
| - | #Add X-gal( | + | #Add X-gal(40μL) and ampicillin(10μL)[200μg/mL] on LB agar medium(IPTG) |
| - | #Add one incubated( | + | #Add one incubated(100μL) |
#Cultivation(overnight) | #Cultivation(overnight) | ||
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===Colony PCR=== | ===Colony PCR=== | ||
Reagent | Reagent | ||
| - | :*TaKaRa Ex Taq(5units/ | + | :*TaKaRa Ex Taq(5units/μL) 0.5μL |
| - | :*10×Ex Taq buffer | + | :*10×Ex Taq buffer 10μL |
| - | :*dNTP Mixture(2.5Meach) | + | :*dNTP Mixture(2.5Meach) 8μL |
| - | :*Primer F( | + | :*Primer F(10μM) 4μL |
| - | :*Primer R( | + | :*Primer R(10μM) 4μL |
:*Template(E.coli DH5α) | :*Template(E.coli DH5α) | ||
| - | :*sterilized water(73. | + | :*sterilized water(73.5μL) |
Conditions of the thermal cycler | Conditions of the thermal cycler | ||
#95°C(5min) | #95°C(5min) | ||
| Line 159: | Line 159: | ||
===Colony PCR=== | ===Colony PCR=== | ||
Reagent | Reagent | ||
| - | :*TaKaRa Ex Taq(5units/ | + | :*TaKaRa Ex Taq(5units/μL) 0.5μL |
| - | :*10×Ex Taq buffer | + | :*10×Ex Taq buffer 10μL |
| - | :*dNTP Mixture(2.5Meach) | + | :*dNTP Mixture(2.5Meach) 8μL |
| - | :*Primer F( | + | :*Primer F(10μM) 4μL |
| - | :*Primer R( | + | :*Primer R(10μM) 4μL |
:*Template(E.coli DH5α) | :*Template(E.coli DH5α) | ||
| - | :*sterilized water(73. | + | :*sterilized water(73.5μL) |
Conditions of the thermal cycler | Conditions of the thermal cycler | ||
#95°C(5min) | #95°C(5min) | ||
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#Centrifuge(6,000rpm,5min,4°C) | #Centrifuge(6,000rpm,5min,4°C) | ||
#Add 5mMEDTA 1mL | #Add 5mMEDTA 1mL | ||
| - | #Add 10%SDS | + | #Add 10%SDS 100μL |
| - | #Add proteinase K | + | #Add proteinase K 50methodL |
#Vortex | #Vortex | ||
#Incubation(30min,55°C) | #Incubation(30min,55°C) | ||
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#Pick up supernatant,remove new microtube | #Pick up supernatant,remove new microtube | ||
#Repeat step 7-11 | #Repeat step 7-11 | ||
| - | #Add 3M-sodium acetate | + | #Add 3M-sodium acetate 40μL,chilled isopropanol 400μL |
#Vortex | #Vortex | ||
#Wind the DNA by a thin glass rod. | #Wind the DNA by a thin glass rod. | ||
| - | #Rinse chilled 70%-ethanol( | + | #Rinse chilled 70%-ethanol(500μL) |
#Pick up DNA,air dry | #Pick up DNA,air dry | ||
| - | #Add TE buffer | + | #Add TE buffer 500μL |
| - | #Add RNase A | + | #Add RNase A 50μL |
#Incubation(20min,37°C) | #Incubation(20min,37°C) | ||
| - | #Add proteinase K | + | #Add proteinase K 50μL |
#Incubation(1h,37°C) | #Incubation(1h,37°C) | ||
#Add phenol mixture | #Add phenol mixture | ||
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#Centrifuge(16,000rpm,10min,4°C) | #Centrifuge(16,000rpm,10min,4°C) | ||
#Pick up supernatant,remove new microtube | #Pick up supernatant,remove new microtube | ||
| - | #Add 3M-sodium acetate | + | #Add 3M-sodium acetate 40μL,chilled isopropanol 400μL |
#Wind the DNA by a thin glass rod. | #Wind the DNA by a thin glass rod. | ||
| - | #Rinse chilled 70%-ethanol( | + | #Rinse chilled 70%-ethanol(500μL,about 30s) |
#Pick up DNA,air dry | #Pick up DNA,air dry | ||
| - | #Add TE buffer | + | #Add TE buffer 200μL |
#*Melt DNA in buffer | #*Melt DNA in buffer | ||
|} | |} | ||
Revision as of 03:40, 22 August 2012
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Editing now.
Creating parts of Tar methylation regionDate:8/9Colony PCRReagent
Conditions of the thermal cycler
Date:8/11The purified DNA
PCR Product
Date:8/13Confirmed of electrophoresis by PCR product and Ligation of the TA vector
Date:8/14Transformation
Creating parts of azurinDate:8/16Colony PCRReagent
Conditions of the thermal cycler
LigationReagent
Method
Date:8/18Colony PCRReagent
Conditions of the thermal cycler
Date:8/20DNA extraction and purification of P.aeruginosa
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