Template:Kyoto/Notebook/Sep 14

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Contents

Sep 14

Restriction Enzyme Digestion

No name

8/22 psB1C3-(2)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts8µL0.5µL0.5µL0µL0µL3µL0µL0.3µL17.7µL30µL
NC1µL0µL0µL0µL0µL1µL0µL0.1µL7.9µL10µL
2 cuts8µL0µL0µL0.5µL0.5µL0µL3µL0.3µL17.7µL30µL
NC1µL0µL0µL0µL0µL0µL1µL0.1µL7.9µL10µL
8/21 tRMA-spinach(1)XbaISpeIBufferBBSAMilliQtotal
2 cuts4µL0.5µL0.5µL3µL0.3µL21.7µL30µL
NC0.5µL0µL0µL1µL0.1µL8.4µL10µL
8/21 tetR aptamer12-1R(1)EcoRISpeIBufferBBSAMilliQtotal
2 cuts3µL0.5µL0.5µL3µL0.3µL22.7µL30µL
NC0.4µL0µL0µL1µL0.1µL8.5µL10µL
8/21 PT181 attenuator-(2)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts4µL0.5µL0.5µL0µL0µL3µL0µL0.3µL21.7µL30µL
NC0.5µL0µL0µL0µL0µL1µL0µL0.1µL8.4µL10µL
2 cuts4µL0µL0µL0.5µL0.5µL0µL3µL0.3µL21.7µL30µL
NC0.5µL0µL0µL0µL0µL0µL1µL0.1µL8.4µL10µL
8/21 PT181 antisense-(2)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts10µL0.5µL0.5µL0µL0µL3µL0µL0.3µL15.7µL30µL
NC1µL0µL0µL0µL0µL1µL0µL0.1µL7.9µL10µL
2 cuts10µL0µL0µL0.5µL0.5µL0µL3µL0.3µL15.7µL30µL
NC1µL0µL0µL0µL0µL0µL1µL0.1µL7.9µL10µL
  • incubated 37°C 1hour

Igku Aug29 Restriction Enzyme Digestion(N③).jpg

Liquid Culture

No name

Samplemedium
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1)Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(2)Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(3)Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(4)Plusgrow medium(+Amp)
8/29 Plux-RBS-GFP-DT-(1)Plusgrow medium(+CP)
8/29 Plux-RBS-GFP-DT-(2)Plusgrow medium(+CP)
8/29 Pbad/araC-RBS-RFP-DT-(1)Plusgrow medium(+CP)
8/29 Pbad-araC-RBS-RFP-DT-(2)Plusgrow medium(+CP)
RBS-lysis3-DT-(1)Plusgrow medium(+CP)

PCR

No name

genome DNAKOD plus10x bufferdNTPMgSO4SasA_fwd primerSasA_rev primerMilliQtotal
0.52.52.51.50.750.75 25
PreDenatureDenatureAnnealingExtensioncycle
　°C　°C　°C　°C--
（◎Д◎）　　　　

Restriction Enzyme Digestion

No name

8/22 psB1C3-(2)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts8µL0.5µL0.5µL0µL0µL3µL0µL0.3µL17.7µL30µL
NC1µL0µL0µL0µL0µL1µL0µL0.1µL7.9µL10µL
2 cuts8µL0µL0µL0.5µL0.5µL0µL3µL0.3µL17.7µL30µL
NC1µL0µL0µL0µL0µL0µL1µL0.1µL7.9µL10µL
8/21 tRMA-spinach(1)XbaISpeIBufferBBSAMilliQtotal
2 cuts4µL0.5µL0.5µL3µL0.3µL21.7µL30µL
NC0.5µL0µL0µL1µL0.1µL8.4µL10µL
8/21 tetR aptamer12-1R(1)EcoRISpeIBufferBBSAMilliQtotal
2 cuts3µL0.5µL0.5µL3µL0.3µL22.7µL30µL
NC0.4µL0µL0µL1µL0.1µL8.5µL10µL
8/21 PT181 attenuator-(2)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts4µL0.5µL0.5µL0µL0µL3µL0µL0.3µL21.7µL30µL
NC0.5µL0µL0µL0µL0µL1µL0µL0.1µL8.4µL10µL
2 cuts4µL0µL0µL0.5µL0.5µL0µL3µL0.3µL21.7µL30µL
NC0.5µL0µL0µL0µL0µL0µL1µL0.1µL8.4µL10µL
8/21 PT181 antisense-(2)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts10µL0.5µL0.5µL0µL0µL3µL0µL0.3µL15.7µL30µL
NC1µL0µL0&;L0µL0µL1µL0µL0.1µL7.9µL10µL
2 cuts10µL0µL0µL0.5µL0.5µL0µL3µL0.3µL15.7µL30µL
NC1µL0µL0µL0µL0µL0µL1µL0.1µL7.9µL10µL
  • incubated 37°C 1hour

Igku Aug29 Restriction Enzyme Digestion(N③).jpg


Ligation

Nakamoto and Hirano

stateVectorInserterLigation High ver.2
experiment8/30 pSB1C3 (EcoRI & SpeI)6.68/30 tRNA-Spinach (EcoRI & SpeI)12.53.5
experiment8/30 pSB1C3 (EcoRI & SpeI)6.68/30 pT181 antisense (EcoRI & SpeI)12.53.5
experiment8/30 pSB1C3 (XbaI & Pst)12.58/30 pT181 attenuator (XbaI & PstI)253.5
experiment8/30 pSB1C3 (XbaI & Pst)12.58/30 pT181 antisense (XbaI & PstI)253.5
experiment8/20 DT (EcoRI & XbaI)6.68/30 pT181 attenuator(2) (EcoRI & SpeI)12.53.5
experiment8/20 DT (EcoRI & XbaI)6.68/30 pT181 antisense (EcoRI & SpeI)12.53.5
experiment8/27 DT (EcoRI & XbaI)3.08/30 tRNA-Spinach (EcoRI & SpeI)12.53.5
experiment8/20 DT (EcoRI & XbaI)6.68/30 RBS-lysis2 (EcoRI & SpeI)12.53.5
experiment8/22 J23100 (SpeI & PstI)--RBS-tetR-DT (XbaI & PstI)--3.5
experiment8/22 Plac (SpeI & PstI)8.08/18 RBS-lacZα-DT (XbaI & PstI)103.5

Samples were evaporeted used evaporator into about 7 µL


Colony PCR

No name

Samplebase pair
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT(8/20 LB Amp) (1)~(4)2143
8/29 Plux-RBS-GFP-DT(8/27 LB-CP) (1)~(2)1227
8/29 Pbad/araC-RBS-RFP{BBa_I13516} (8/21 LB-CP) (1)~(2)2256
8/29 RBS-lysis3-DT-(1)1210
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s2min30cycle

Liquid Culture

No name

Samplemedium
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1)Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(2)Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(3)Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(4)Plusgrow medium(+Amp)
8/29 Plux-RBS-GFP-DT-(1)Plusgrow medium(+CP)
8/29 Plux-RBS-GFP-DT-(2)Plusgrow medium(+CP)
8/29 Pbad/araC-RBS-RFP-DT-(1)Plusgrow medium(+CP)
8/29 Pbad-araC-RBS-RFP-DT-(2)Plusgrow medium(+CP)
RBS-lysis3-DT-(1)Plusgrow medium(+CP)

Transformation

No Name

NameSampleCompetent CellsTotalPlate
8/27 pT181 attenuator-DT2µL20µL22µLCP
8/27 RBS-lysis3-DT2µL20µL22µLCP
8/28 RBS-lysis3-DT2µL20µL22µLCP
8/28 Pcon-RBS-GFP-DT-Pcon-RBS-LuxR-DT2µL20µL22µLAmp
8/28 RBS-lysis1-DT2µL20µL22µLCP
8/28 Plux-RBS-GFP-DT2µL20µL22µLCP
8/28 Spinach-DT2µL20µL22µLCP

Igku Aug29 Transformation1(N④-1).jpg
Igku Aug29 Transformation(N④-2).jpg
Igku Aug29 Transformation(N⑤).jpg
File:Igku Aug29 Transformation()after


PCR

No name

genome DNAKOD plus10x bufferdNTPMgSO4SasA_fwd primerSasA_rev primerMilliQtotal
0.52.52.51.50.750.75 25
PreDenatureDenatureAnnealingExtensioncycle
　°C　°C　°C　°C--
（◎Д◎）　　　　
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