Template:Kyoto/Notebook/Sep 22

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Contents

Sep 22

Colony PCR

Tatsui

Samplebase pair
9/20 Plac-PT181 attenuator(1~4)664
9/20 Plac-PT181 antisense(1~6)369
9/20 Pcon-PT181 attenuator-aptamer-DT(1~6)859
9/20 Pcon-PT181 antisense-spinach-DT(1~8)723

Miniprep

Honda

DNAconcentration[µg/mL]260/280260/230
9/21 spinach-DT197.91.671.54
9/21 Plux125.91.711.27
9/21 Plux-RBS-GFP-DT-(1)168.01.621.73
9/21 Pcon-PT181 attenuator-(1)368.31.571.62
9/21 RBS-GFP-DT532.81.751.78
9/21 Pcon-RBS-GFP-DT845.22.021.70

Colony PCR

Honda

Samplebase pair
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1~2)283
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s2min10s30cycles

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2~4Pcon-aptamer12-1R-DT(E-1A)--
6~8Pcon-spinach-DT(E-1A)--
10~12Pcon-attenuator PT181(E-1A)--
LaneDNAEnzyme
1100bp ladder--
2~4DT(1-3)--
6~8aptamer12-1RDT(1-3)--
10~12Pcon-PT181 attenuator(1-5A)--
Nameconcentration[µg/mL]260/280260/230
Pcon-aptamer12-1R-DT(E-1A)31.21.870.30
Pcon-spinach-DT(E-1A)25.61.880.99
Pcon-attenuator PT181(E-1A)25.31.990.86
DT(1-3)22.11.820.20
aptamer12-1RDT(1-3)33.51.990.05
Pcon-PT181 attenuator(1-5A)36.91.120.96

digestion

No name

9/16 PlacXbaISpeIPstIBufferBBSAMilliQtotal
2cuts12.5µL0µL1µL1µL3µL3µL9.5µL30µL
NC0.8µL0µL0µL0µL1µL1µL7.4µL10µL
9/22 PluxXbaISpeIPstIBufferBBSAMilliQtotal
2cuts16µL0µL1µL1µL3µL3µL14µL30µL
NC0.8µL0µL0µL0µL1µL1µL7.2µL10µL
9/20 RBS-tetR-2R-DT XbaISpeIPstIBufferDBSAMilliQtotal
2cuts8.5µL1µL0µL1µL3µL3µL13.5µL30µL
NC0.4µL0µL0µL0µL1µL1µL7.6µL10µL

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2~4Pcon-attenuator PT181(E-0)--
LaneDNAEnzyme
2100bp ladder--
4~6Pcon-attenuator PT181(2-SA)--
LaneDNAEnzyme
1100bp ladder--
3~5spinach-DT(3-2)--
8~10Ptet-antisense PT181(1-3)--
Nameconcentration[µg/mL]260/280260/230
Pcon-attenuator PT181(E-0)2.41.800.17
Pcon-attenuator PT181(2-SA)10.81.850.54
spinach-DT(3-2)36.71.800.75
Ptet-antisense PT181(1-3)16.81.740.78

Colony PCR

No name

Samplebase pair
9/20 Pcon-PT181 attenuator-aptamer12-1R-DT-(7~14)859

Colony PCR

Tatsui

Samplebase pair
PconPT181antisense-spinach-DT(9~15)723


Ligation

Okazaki, Kanaoka, Tatsui and Hirano

stateVectorInserterLigation High ver.2
experiment9/18 Plac (SpeI & PstI)3.0 µL9/18 RBS-lysis3-DT (XbaI & PstI)12.4 µL3.5 µL
experiment9/12 Ptet-pT181 antisense1.0 µL9/18 spinach-DT11.9 µL3.5 µL
experiment9/13 Pcon-RBS-luxR-DT2.1 µL9/10 Plux-RBS-GFP-DT5.6 µL3.5 µL
experiment9/13 Pcon-pT181 attenuator (SpeI & PstI)2.0 µL9/18 aptamer 12-1R-DT (XbaI & PstI)6.3 µL3.5 µL
experiment9/18 Plac ②(SpeI & PstI)3.0 µL9/18 aptamer 12-1R-DT(XbaI & PstI)4.5µLx3.5 µL

Liquid Culture

No name

Samplemedium
9/2 spinach-DT-2--
8/16 Plux-4--
8/27 Plux-RBS-GFP-DT-1--
9/5 Pcon-pT181 attenuator-1--

digestion

Gel Extraction

Nakamoto

LaneDNAEnzyme
1100bp ladder--
3RBS-lacZα-DTXbaI&PstI
4RBS-lacZα-DTXbaI&PstI
5RBS-lacZα-DTXbaI&PstI
6RBS-lacZα-DTXbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(XbaI&PstI)9.81.500.39


Transformation

Tatsui

NameSampleCompetent CellsPlate
9/17 pSB1C3-Pcon-pT181 attenuator-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-aptamer 12_1R-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-antisense-spinach-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-RBS-tetR-DT x22 µL20 µLCP
9/14 pSB1C3-PkaiBC2 µL20 µLCP
9/14 pSB1C3-RpaB2 µL20 µLCP
9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI) 2 µL20 µLCP
9/17 pSB1C3-pT181 attenuator(XbaI&PstI) 2 µL20 µLCP
9/17 pSB1C3-Pcon-spinach-DT2 µL20 µLCP
9/14 RBS-GFP-DT-PkaiBC2 µL20 µLCP
Plux-RBS-lysis3-DT2 µL20 µLCP
9/20 Ptet-pT181 antisense-spinach-DT2 µL20 µLCP
9/20 Pcon-spinach-DT(pSB4K5) 2 µL20 µLKan
9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5) 2 µL20 µLKan
9/20 KaiABC2 µL20 µLKan
9/20 KaiABC2 µL20 µLKan
9/20 Plac-RBS-lysis3-DT2 µL20 µLAmp
9/20 Plac-aptamer 12_1R2 µL20 µLAmp
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT2 µL20 µLAmp
Plac-aptamer12_1R-DT2 µL20 µLAmp
9/20 Plac-aptamer12_1R-DT2 µL20 µLAmp
9/20Pcon-pT181attenuator-aptamer-DT2 µL20 µLAmp
9/17 Plac-pT181-attenuatorx22 µL20 µLAmp
9/15 Pcon-RBS-lacZα-DT2 µL20 µLAmp
9/14 Pcon-pT181attenuator-RBS-lacZα-DT2 µL20 µLAmp
PUC192 µL20 µLAmp

PCR

Tatsui

Samplebase pair
pT181 attenuator(pSB1C3)--
Pcon-RBS-lacZα-DT--
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C55 °C68 °C--
5 min30 s30 s3 min30 cycles


Transformation

Tatsui

NameSampleCompetent CellsPlate
9/17 pSB1C3-Pcon-pT181 attenuator-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-aptamer 12_1R-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-antisense-spinach-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-RBS-tetR-DT x22 µL20 µLCP
9/14 pSB1C3-PkaiBC2 µL20 µLCP
9/14 pSB1C3-RpaB2 µL20 µLCP
9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI) 2 µL20 µLCP
9/17 pSB1C3-pT181 attenuator(XbaI&PstI) 2 µL20 µLCP
9/17 pSB1C3-Pcon-spinach-DT2 µL20 µLCP
9/14 RBS-GFP-DT-PkaiBC2 µL20 µLCP
Plux-RBS-lysis3-DT2 µL20 µLCP
9/20 Ptet-pT181 antisense-spinach-DT2 µL20 µLCP
9/20 Pcon-spinach-DT(pSB4K5) 2 µL20 µLKan
9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5) 2 µL20 µLKan
9/20 KaiABC2 µL20 µLKan
9/20 KaiABC2 µL20 µLKan
9/20 Plac-RBS-lysis3-DT2 µL20 µLAmp
9/20 Plac-aptamer 12_1R2 µL20 µLAmp
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT2 µL20 µLAmp
Plac-aptamer12_1R-DT2 µL20 µLAmp
9/20 Plac-aptamer12_1R-DT2 µL20 µLAmp
9/20Pcon-pT181attenuator-aptamer-DT2 µL20 µLAmp
9/17 Plac-pT181-attenuatorx22 µL20 µLAmp
9/15 Pcon-RBS-lacZα-DT2 µL20 µLAmp
9/14 Pcon-pT181attenuator-RBS-lacZα-DT2 µL20 µLAmp
PUC192 µL20 µLAmp