Template:Kyoto/Notebook/Sep 11

From 2013.igem.org

Contents

Sep 11

Electrophoresis

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

Igku 9111.jpg

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

Igku 9112.jpg

colony PCR

Tatsui

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

Tatsui

PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5min30s　30s　42s　30


Samplebase pair
Pbad\araC-RBS-luxI-DT
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5min30s　30s　2min　30

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

Igku 9113.jpg

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

Igku 9114.jpg

colony PCR

Samplebase pair
9/9Pcon+RBS-luxI-DT1079bp
9/10Pcon+sptamer12_1R-DT584bp
9/10DT+Pcon-pT181 attenuator 1781bp
9/10Fusion1 attenuator(p3B1C3)-1638bp
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
50sec　30sec　30sec　1min　30

Gel Extraction

Igku 9115.jpg
Igku 9116.jpg
Igku 9117.jpg
Igku 9118.jpg

Ligation

Okazaki

stateVectorInserterLigation High ver.2
experiment9/8pSB4K5(EcoRI&SpeI)21.8ng/µL2.3 µL9/8Pcon-Spinach-DT(EcoRI&SpeI)10.5ng/µL1.7 µL2.0 µL
experment9/8pSB4K5(EcoRI&SpeI)21.8ng/µL2.3 µL9/8RBS-lacZα-DT(EcoRI&SpeI)4.4ng/µL 6.7µL3.5 µL
experiment9/8pSB4K5(EcoRI&SpeI)21.8ng/µL2.3 µL9/8Pcon-RBS-lacZα-DT(EcoRI&SpeI)5.6ng/µL1.9 µL2.1 µL
experiment9/6Plux 25ng/µL2 µL9/8 RBS-lysis1-DT(XbaI&PstI)6.6 µL3.5 µL
experiment9/6Plux 25ng/µL2 µL9/8 RBS-lysis3-DT(XbaI&PstI)4.7 µL3.4 µL
experiment9/10Pcon-pT181 attenuator(SpeI&PstI) 3.4µL9/8RBS-lacZα-DT(EcoRI&SpeI)4.5 µL3.5 µL
experiment9/6Pcon-RBS laxR-DT(EcoRI&XbaI) 26.7ng/µL1.9 µLPlax-RBS-GFP-DT5.5 µL3.5 µL
experiment9/9 Pcon-pT181 attenuator(SpeI&PstI)3.4 µL9/10 aptamer12_1R-DT(XbaI&PstI)1.7 µL2.6 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 attenuator(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-pT181attenuator+RBS-lacZα-DT2µL20µL22µLAmp
Pcon-iuxR+Plux-GFP2µL20µL22µLAmp
Pcon-pT181attenuator+apt12_1R-DT2µL20µL22µLAmp
Plac(BBa_R0011)2µL20µL22µLAmp

Electrophoresis

Tatsui

LaneSample
1100bp ladder
2Pcon+RBS luxI-DT2
3Pcon+RBS luxI-DT3
4Pcon+RBS luxI-DT4
5Pcon+RBS luxI-DT5
6Pcon+RBS luxI-DT6
7Pcon+apF12_1R-DT1
8Pcon+apF12_1R-DT2
9Pcon+apF12_1R-DT3
10Pcon+apF12_1R-DT4
11DT+Pcon-pT181attenuator1
12F1attenuator(pSB1C3)
13100bp ladder

Igku 9119.jpg

Restriction Enzyme Digestion

Nakamoto

DNAEcoRIXbaISpeIPstIbufferBSAMilliQTotal
pSB4K5(EcoRI+SpeI)9.4µL 1µL 0µL 1µL 0µL 3µL 3µL 12.6µL30 µL
pSB4K5(EcoRI+SpeI) negative0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
RBS-lacZα-DT(EcoRI+SpeI) 10µL 1µL 0µL 1µL 0µL 3µL 3µL 12µL 30µL
RBS-lacZα-DT(EcoRI+SpeI) negative 0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
Ptet-pT181 antisense(SteI+PstI) 10.8µL 0µL 0µL 1µL 1µL 3µL 3µL 12µL 30µL
Ptet-pT181 antisense(SteI+PstI) negative 0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
Pcon-RBS-lacZα-DT(EcoRI+SpeI) 7.1µL 1µL 0µL 1µL 0µL 3µL 3µL 14.9µL 30µL
Pcon-RBS-lacZα-DT(EcoRI+SpeI) negative 0.4µL 0µL 0µL 0µL 0µL 1µL 1µL 7.6µL 10µL
RBS-lysis2-DT(XbaI+PstI) 22µL 0µL 1µL 0µL 1µL 3µL 3µL 0µL 30µL
RBS-lysis2-DT(XbaI+PstI) 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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