Template:Kyoto/Notebook/Sep 11

From 2013.igem.org

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(→Electrophoresis)
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|9/9Pcon-spinach-DT(p3B4k5)||605bp
|9/9Pcon-spinach-DT(p3B4k5)||605bp
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===Electrophoresis===
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<!-- こっから -->
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<div class="experiment">
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<span class="author">Tatsui</span>
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{| class="wikitable"
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!Lane||Sample
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|-
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|1||100bp ladder 
 +
|-
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|2||Pcon-lacZ&alpha;(pSB4K5)3
 +
|-
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|3||Pcon-lacZ&alpha;(pSB4K5)4 
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|-
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|4||Pcon-lacZ&alpha;(pSB4K5)5
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|-
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|5||Pcon-lacZ&alpha;(pSB4K5)6
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|-
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|6||Pcon-spinach-DT(pSB4K5)3
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|-
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|7||Pcon-spinach-DT(pSB4K5)4 
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|-
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|8||Pcon-spinach-DT(pSB4K5)5 
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|-
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|9||Pcon-spinach-DT(pSB4K5)6 
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|-
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|10||100bp ladder
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|}
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[[File:igku_xxxxxx.xxx]]<br>
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</div>
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{| class="wikitable"
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!Lane||Sample
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|-
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|1||1kbp ladder 
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|-
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|2||Pbad/araC-RBS-luxI-DT3
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|-
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|3||Pbad/araC-RBS-luxI-DT4 
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|-
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|4||Pbad/araC-RBS-luxI-DT5
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|-
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|5||Pbad/araC-RBS-luxI-DT6
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|-
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|6||1kbp ladder
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|}
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[[File:igku_xxxxxx.xxx]]<br>
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</div>
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 +
<div class="experiment">
 +
<span class="author">Tatsui</span>
{| class="wikitable"
{| class="wikitable"
!PreDenature||Denature||Annealing||Extension||cycle
!PreDenature||Denature||Annealing||Extension||cycle

Revision as of 16:47, 24 September 2013

Contents

Sep 11

Electrophoresis

LaneSample
11kladder
2Pbad/araC+RBS-luxI-DT-1
3Pbad/araC+RBS-luxI-DT-1
4Plac+RBS-lysis-DT
5Pcon+RBS-luxI-DT
61kladder

File:Igku xxxxxx.xxx

PCR

Tatsui

pKaiBC(200p)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
0.52.52.51.51.50.51625
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　
pKaiBC(400p)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
12.52.51.51.50.515.525
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　
pKaiBC(800p)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
22.52.51.51.50.514.525
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　
pKaiBC(1ng)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
2.52.52.51.51.50.51425
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　

Electrophoresis

Tatsui

LaneSample
1100bp ladder
2pKaiBC 800pg 60°C
3pKaiBC 1ng 60°C
4pKaiBC 800pg 61°C
5pKaiBC 1ng 61°C
6pKaiBC 800pg 62°C
7pKaiBC 1ng 62°C
8100bp ladder

File:Igku xxxxxx.xxx

colony PCR

Tatsui

Samplebase pair
9/9Pcon-lacZα(p3B4k5)712bp
9/9Pcon-spinach-DT(p3B4k5)605bp

Electrophoresis

Tatsui

LaneSample
1100bp ladder
2Pcon-lacZα(pSB4K5)3
3Pcon-lacZα(pSB4K5)4
4Pcon-lacZα(pSB4K5)5
5Pcon-lacZα(pSB4K5)6
6Pcon-spinach-DT(pSB4K5)3
7Pcon-spinach-DT(pSB4K5)4
8Pcon-spinach-DT(pSB4K5)5
9Pcon-spinach-DT(pSB4K5)6
10100bp ladder

File:Igku xxxxxx.xxx

LaneSample
11kbp ladder
2Pbad/araC-RBS-luxI-DT3
3Pbad/araC-RBS-luxI-DT4
4Pbad/araC-RBS-luxI-DT5
5Pbad/araC-RBS-luxI-DT6
61kbp ladder

File:Igku xxxxxx.xxx

Tatsui

PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5min30s　30s　42s　
Samplebase pair
Pbad\araC-RBS-luxI-DT
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5min30s　30s　2min　
Samplebase pair
9/9Pcon+RBS-luxI-DT1079bp
9/10Pcon+sptamer12_1R-DT584bp
9/10DT+Pcon-pT181 attenuater 1781bp
9/10Fusion1 attenuater(p3B1C3)-1638bp
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
50sec　30sec　30sec　1min　

Ligation

Okazaki

stateVectorInserterLigation High ver.2
9/8pSB4K5(EcoRl&Spel)21.8ng/µL2.3 µL9/8Pcon-Spinach-DT(EcoRl&Spel)10.5ng/µL1.7 µL4.0 µL
9/8pSB4K5(EcoRl&Spel)21.8ng/µL2.3 µL9/8RBS-lacZα-DT(EcoRl&Spel)4.4ng/µL 6.7µL9.0 µL
9/8pSB4K5(EcoRl&Spel)21.8ng/µL2.3 µL9/8Pcon-RBS-lacZα-DT(EcoRl&Spel)5.6ng/µL1.9 µL4.2 µL
9/6Plux 25ng/µL2 µL9/8 RBS-lysis1-DT(Xbal&Pstl)6.6 µL8.6 µL
9/6Plux 25ng/µL2 µL9/8 RBS-lysis3-DT(Xbal&Pstl)4.7 µL6.7 µL
9/10Pcon-pT181 attenuater(Spel&Pstl) 3.4µL9/8RBS-lacZα-DT(EcoRl&Spel)4.5 µL7.9 µL
9/6Pcon-RBS laxR-DT(EcoRl&Xbal) 26.7ng/µL1.9 µLPlax-RBS-GFP-DT5.5 µL7.4 µL
9/9 Pcon-pT181 attenuater(SPpel&Pstl)3.4 µL9/10 aptamer12_1R-DT(Xbal&Pstl)1.7 µL5.1 µL

Transformation

Nakamoto Tatsui

NameSampleCompetent CellsTotalPlate
9/9 Fusion1 antisense(pSB2C3)2µL20µL22µLCP
9/9 Fusion6 antisense(pSB2C3)2µL20µL22µLCP
9/9 aptamer 12_P(pSB2C3)2µL20µL22µLCP
9/9 aptamer 12_1&micro(pSB2C3)2µL20µL22µLCP
9/9 Fusion2 attenuater(pSB2C3) 2µL20µL22µLCP
9/11 Pcon-spinach-DT(pSB4K5)2µL20µL22µLK
9/11 RBS-lacZα-DT(pSB4K5)2µL20µL22µLK
9/11 Pcon-RBS-lacZα-DT(pSB4K5)2µL20µL22µLK
Plux+RBS-lysis1-DT2µL20µL22µLCP
Plux+RBS-lysis3-DT2µL20µL22µLCP
Pcon-pT181attenuater+RBS-lacZα-DT2µL20µL22µLAmp
Pcon-iuxR+Plux-GFP2µL20µL22µLAmp
Pcon-pT181attenuater+apt12_1R-DT2µL20µL22µLAmp
Plac(BBa_R0011)2µL20µL22µLAmp

Restriction Enzyme Digestion

Nakamoto

DNAEcoRlXbalSpelPstlbufferBSAMilliQTotal
pSB4K5(EcoRl+Spel)9.4µL 1µL 0µL 1µL 0µL 3µL 3µL 12.6µL30 µL
pSB4K5(EcoRl+Spel) negative0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
RBS-lacZα-DT(EcoRl+Spel) 10µL 1µL 0µL 1µL 0µL 3µL 3µL 12µL 30µL
RBS-lacZα-DT(EcoRl+Spel) negative 0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
Ptet-pT181 antisense(Stel+Pstl) 10.8µL 0µL 0µL 1µL 1µL 3µL 3µL 12µL 30µL
Ptet-pT181 antisense(Stel+Pstl) negative 0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
Pcon-RBS-lacZα-DT(EcoRl+Spel) 7.1µL 1µL 0µL 1µL 0µL 3µL 3µL 14.9µL 30µL
Pcon-RBS-lacZα-DT(EcoRl+Spel) negative 0.4µL 0µL 0µL 0µL 0µL 1µL 1µL 7.6µL 10µL
RBS-lysis2-DT(Xbal+Pstl) 22µL 0µL 1µL 0µL 1µL 3µL 3µL 0µL 30µL
RBS-lysis2-DT(Xbal+Pstl) negative 1.4µL 0µL 0µL 0µL 0µL 1µL 1µL 6.6µL 10µL

Liquid Culture

Nakamoto

Samplemedium
8/16 Master4 Plax
8/25 Pλ-laxI-2 　

PCR

Hirano

8/29 Pλ-RBS-luxI-DT(76.5ng/µL)10x buffer for KOD plus-ver.2KOD-plusdNTPMgSO4F primer(RBS-luxI-DT-cloning-fwd)R primer(RBS-luxI-DT-cloning-rev)MilliQtotal
0.32.50.52.51.50.750.7516.225
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　65°C　68°C--
5min30sec30sec1min30
8/31 Pbad/araC-RBS-RFP(1)(67.6ng/µL)10x buffer for KOD plus-ver.2KOD-plusdNTPMgSO4F primer(Pbad/araC(pSBlc3)-cloning-fwd)R primer(Pbad/araC(pSBlc3)-cloning-rev)MilliQtotal
0.32.50.52.51.50.750.7516.225
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　54°C　68°C--
5min30sec30sec3min 20sec30

Liquid Culture

Hirano

Samplemedium
9/4 RBS-lysis1+DT 3plusgrow(+CP) 3ml 37°C
9/4 RBS-lysis2+DT 1plusgrow(+CP) 3ml 37°C
9/4 RBS-lysis3+DT 3plusgrow(+CP) 3ml 37°C
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