Template:Kyoto/Notebook/Sep 11

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Contents

Sep 11

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 

colony PCR

Tatsui

Samplebase pair
9/9Pcon-lacZα(p3B4k5)712bp
9/9Pcon-spinach-DT(p3B4k5)605bp
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
experiment2.3 µLPcon-Spinach-DT(EcoRl&Spel)10.5ng/µL1.7 µL4.0 µL
NC9/8pSB4K5(EcoRl&Spel)21.8ng/µLxx µLnamexx µLxx µL
NCnamexx µLnamexx µLxx µL
NCnamexx µLnamexx µLxx µL
NCnamexx µLnamexx µLxx µL
NCnamexx µLnamexx µLxx µL
NCnamexx µLnamexx µLxx µL
NCnamexx µLnamexx µLxx µL

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