From 2013.igem.org
Sep 11
Electrophoresis
| Lane | Sample
|
| 1 | 1kladder
|
| 2 | Pbad/araC+RBS-luxI-DT-1
|
| 3 | Pbad/araC+RBS-luxI-DT-1
|
| 4 | Plac+RBS-lysis-DT
|
| 5 | Pcon+RBS-luxI-DT
|
| 6 | 1kladder
|
PCR
Tatsui
| pKaiBC(200p) | 10*buffer KOD plus | dNTPs | MgSO4 | primer mix(F&R) | KOD plus | MilliQ | total
|
| 0.5 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 16 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 60°C | 68°C | 30
|
| 2min | 10sec | 30sec | 30sec |
|
| pKaiBC(400p) | 10*buffer KOD plus | dNTPs | MgSO4 | primer mix(F&R) | KOD plus | MilliQ | total
|
| 1 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 15.5 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 60°C | 68°C | 30
|
| 2min | 10sec | 30sec | 30sec |
|
| pKaiBC(800p) | 10*buffer KOD plus | dNTPs | MgSO4 | primer mix(F&R) | KOD plus | MilliQ | total
|
| 2 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14.5 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 60°C | 68°C | 30
|
| 2min | 10sec | 30sec | 30sec |
|
| pKaiBC(1ng) | 10*buffer KOD plus | dNTPs | MgSO4 | primer mix(F&R) | KOD plus | MilliQ | total
|
| 2.5 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 60°C | 68°C | 30
|
| 2min | 10sec | 30sec | 30sec |
|
Electrophoresis
Tatsui
| Lane | Sample
|
| 1 | 100bp ladder
|
| 2 | pKaiBC 800pg 60°C
|
| 3 | pKaiBC 1ng 60°C
|
| 4 | pKaiBC 800pg 61°C
|
| 5 | pKaiBC 1ng 61°C
|
| 6 | pKaiBC 800pg 62°C
|
| 7 | pKaiBC 1ng 62°C
|
| 8 | 100bp ladder
|
File:Igku xxxxxx.xxx
colony PCR
Tatsui
| Sample | base pair
|
| 9/9Pcon-lacZα(p3B4k5) | 712bp
|
| 9/9Pcon-spinach-DT(p3B4k5) | 605bp
|
Tatsui
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 5min | 30s | 30s | 42s |
|
| Sample | base pair
|
| Pbad\araC-RBS-luxI-DT |
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 5min | 30s | 30s | 2min |
|
Electrophoresis
Tatsui
| Lane | Sample
|
| 1 | 100bp ladder
|
| 2 | Pcon-lacZα(pSB4K5)3
|
| 3 | Pcon-lacZα(pSB4K5)4
|
| 4 | Pcon-lacZα(pSB4K5)5
|
| 5 | Pcon-lacZα(pSB4K5)6
|
| 6 | Pcon-spinach-DT(pSB4K5)3
|
| 7 | Pcon-spinach-DT(pSB4K5)4
|
| 8 | Pcon-spinach-DT(pSB4K5)5
|
| 9 | Pcon-spinach-DT(pSB4K5)6
|
| 10 | 100bp ladder
|
File:Igku xxxxxx.xxx
| Lane | Sample
|
| 1 | 1kbp ladder
|
| 2 | Pbad/araC-RBS-luxI-DT3
|
| 3 | Pbad/araC-RBS-luxI-DT4
|
| 4 | Pbad/araC-RBS-luxI-DT5
|
| 5 | Pbad/araC-RBS-luxI-DT6
|
| 6 | 1kbp ladder
|
File:Igku xxxxxx.xxx
colony PCR
| Sample | base pair
|
| 9/9Pcon+RBS-luxI-DT | 1079bp
|
| 9/10Pcon+sptamer12_1R-DT | 584bp
|
| 9/10DT+Pcon-pT181 attenuator 1 | 781bp
|
| 9/10Fusion1 attenuator(p3B1C3)-1 | 638bp
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 50sec | 30sec | 30sec | 1min |
|
Ligation
Okazaki
| state | Vector | Inserter | Ligation High ver.2
|
| 9/8pSB4K5(EcoRl&Spel)21.8ng/µL | 2.3 µL | 9/8Pcon-Spinach-DT(EcoRl&Spel)10.5ng/µL | 1.7 µL | 4.0 µL
|
| 9/8pSB4K5(EcoRl&Spel)21.8ng/µL | 2.3 µL | 9/8RBS-lacZα-DT(EcoRl&Spel)4.4ng/µL | 6.7µL | 9.0 µL
|
| 9/8pSB4K5(EcoRl&Spel)21.8ng/µL | 2.3 µL | 9/8Pcon-RBS-lacZα-DT(EcoRl&Spel)5.6ng/µL | 1.9 µL | 4.2 µL
|
| 9/6Plux 25ng/µL | 2 µL | 9/8 RBS-lysis1-DT(Xbal&Pstl) | 6.6 µL | 8.6 µL
|
| 9/6Plux 25ng/µL | 2 µL | 9/8 RBS-lysis3-DT(Xbal&Pstl) | 4.7 µL | 6.7 µL
|
| 9/10Pcon-pT181 attenuator(Spel&Pstl) | 3.4µL | 9/8RBS-lacZα-DT(EcoRl&Spel) | 4.5 µL | 7.9 µL
|
| 9/6Pcon-RBS laxR-DT(EcoRl&Xbal) 26.7ng/µL | 1.9 µL | Plax-RBS-GFP-DT | 5.5 µL | 7.4 µL
|
| 9/9 Pcon-pT181 attenuator(SPpel&Pstl) | 3.4 µL | 9/10 aptamer12_1R-DT(Xbal&Pstl) | 1.7 µL | 5.1 µL
|
Transformation
Nakamoto Tatsui
| Name | Sample | Competent Cells | Total | Plate
|
| 9/9 Fusion1 antisense(pSB2C3) | 2µL | 20µL | 22µL | CP
|
| 9/9 Fusion6 antisense(pSB2C3) | 2µL | 20µL | 22µL | CP
|
| 9/9 aptamer 12_P(pSB2C3) | 2µL | 20µL | 22µL | CP
|
| 9/9 aptamer 12_1µ(pSB2C3) | 2µL | 20µL | 22µL | CP
|
| 9/9 Fusion2 attenuator(pSB2C3) | 2µL | 20µL | 22µL | CP
|
| 9/11 Pcon-spinach-DT(pSB4K5) | 2µL | 20µL | 22µL | K
|
| 9/11 RBS-lacZα-DT(pSB4K5) | 2µL | 20µL | 22µL | K
|
| 9/11 Pcon-RBS-lacZα-DT(pSB4K5) | 2µL | 20µL | 22µL | K
|
| Plux+RBS-lysis1-DT | 2µL | 20µL | 22µL | CP
|
| Plux+RBS-lysis3-DT | 2µL | 20µL | 22µL | CP
|
| Pcon-pT181attenuator+RBS-lacZα-DT | 2µL | 20µL | 22µL | Amp
|
| Pcon-iuxR+Plux-GFP | 2µL | 20µL | 22µL | Amp
|
| Pcon-pT181attenuator+apt12_1R-DT | 2µL | 20µL | 22µL | Amp
|
| Plac(BBa_R0011) | 2µL | 20µL | 22µL | Amp |
|
Electrophoresis
Tatsui
| Lane | Sample
|
| 1 | 100bp ladder
|
| 2 | Pcon+RBS luxI-DT2
|
| 3 | Pcon+RBS luxI-DT3
|
| 4 | Pcon+RBS luxI-DT4
|
| 5 | Pcon+RBS luxI-DT5
|
| 6 | Pcon+RBS luxI-DT6
|
| 7 | Pcon+apF12_1R-DT1
|
| 8 | Pcon+apF12_1R-DT2
|
| 9 | Pcon+apF12_1R-DT3
|
| 10 | Pcon+apF12_1R-DT4
|
| 11 | DT+Pcon-pT181attenuator1
|
| 12 | F1attenuator(pSB1C3)
|
| 13 | 100bp ladder
|
File:Igku xxxxxx.xxx
Restriction Enzyme Digestion
Nakamoto
| | DNA | EcoRl | Xbal | Spel | Pstl | buffer | BSA | MilliQ | Total |
|
| pSB4K5(EcoRl+Spel) | 9.4µL | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 12.6µL | 30 µL
|
| pSB4K5(EcoRl+Spel) negative | 0.5µL | 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
| Sample | medium
|
| 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.2 | KOD-plus | dNTP | MgSO4 | F primer(RBS-luxI-DT-cloning-fwd) | R primer(RBS-luxI-DT-cloning-rev) | MilliQ | total
|
| 0.3 | 2.5 | 0.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.2 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 65°C | 68°C | --
|
| 5min | 30sec | 30sec | 1min | 30
|
| 8/31 Pbad/araC-RBS-RFP(1)(67.6ng/µL) | 10x buffer for KOD plus-ver.2 | KOD-plus | dNTP | MgSO4 | F primer(Pbad/araC(pSBlc3)-cloning-fwd) | R primer(Pbad/araC(pSBlc3)-cloning-rev) | MilliQ | total
|
| 0.3 | 2.5 | 0.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.2 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 54°C | 68°C | --
|
| 5min | 30sec | 30sec | 3min 20sec | 30
|
Liquid Culture
Hirano
| Sample | medium
|
| 9/4 RBS-lysis1+DT 3 | plusgrow(+CP) 3ml 37°C
|
| 9/4 RBS-lysis2+DT 1 | plusgrow(+CP) 3ml 37°C
|
| 9/4 RBS-lysis3+DT 3 | plusgrow(+CP) 3ml 37°C
|
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