From 2013.igem.org
Sep 17
Liquid Culture
Tatsui and Hirano
| Sample | medium
|
| Spi-DT | LB medium(+CP)
|
| Pcon-Spi-DT,Pcon-pT181-anti-spi-DT | LB medium(+Amp)
|
PCR
Hirano
| Pcon-tet ap-DT(VF) | BigDye | 5x buffer | primer(VF) | MilliQ | total
|
| 1.3 | 0.5 | 1.75 | 0.5 | 0.45 | 10.5
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 96°C | 96°C | 50°C | 60°C | --
|
| 2m | 10s | 5s | 4m | 30cycles
|
| Pcon-tet ap-DT(VR) | BigDye | 5x buffer | primer(VR) | MilliQ | total
|
| 1.3 | 0.5 | 1.75 | 0.5 | 0.45 | 10.5
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 96°C | 96°C | 50°C | 60°C | --
|
| 2m | 10s | 5s | 4m | 30cycles
|
Electrophoresis
じっけんしゃ
| Lane | Sample | Enzyme1 | Enzyme2
|
| 1 | 1kbpladder | -- | --
|
| 2 | Pcon-RBS-tetR-DT | XbaI | PstI
|
| 3 | Pcon-RBS-tetR-DT | -- | --
|
| 4 | pSB4K5X7 | EcoRI | XbaI
|
| 5 | pSB4K5X7 | -- | --
|
| 6 | PSB1C3 | EcoRI | SpeI
|
| 7 | PSB1C3 | EcoRI | PstI
|
| 8 | PSB1C3 | -- | --
|
| 9 | pT181attenuator | -- | --
|
| 10 | pT181attenuator | -- | --
|
| 11 | 1kbpladder | -- | --
|
File:Igku xxxxxx.xxx
Ligation
No Name
| state | Vector | Inserter | Ligation High ver.2
|
| experiment | 8/28 Plux(SpeI+PstI) | 45µg/mL | 9/13 RBS-lysis1-DT (XbaI+PstI) | 7.8µg/mL | xx µg/mL
|
| experiment | 8/28 Plux(SpeI+PstI) | 45µg/mL | 9/13 RBS-lysis2-DT (XbaI+PstI) | 11.8µg/mL | xx µg/mL
|
| experiment | 8/28 Plux(SpeI+PstI) | 45µg/mL | 9/13 RBS-lysis3-DT (XbaI+PstI) | 11.8µg/mL | xx µg/mL
|
| experiment | 9/14 Plac(SpeI+PstI) | 21µg/mL | 9/16 PT181 atte-DT (XbaI+PstI) | 8.2µg/mL | xx µg/mL
|
| experiment | 9/14 Plac(SpeI+PstI) | 21µg/mL | 9/3 PT181 anti (XbaI+PstI) | 6.6µg/mL | xx µg/mL
|
| experiment | 9/14 Plac(SpeI+PstI) | 21µg/mL | 9/10 apt12-1R-DT(XbaI+PstI) | 15.6µg/mL | xx µg/mL
|
| experiment | 9/13 pSB1C3(XbaI+PstI) | 26.0µg/mL | 9/15 Pcon-PT181 anti-spin-DT(XbaI+PstI) | 5.0µg/mL | xx µg/mL
|
| experiment | 9/16 pSB1C3(EcoRI+SpeI) | 49.5µg/mL | 9/15 Pcon-spin-DT(EcoRI+SpeI) | 9.0µg/mL | xx µg/mL
|
| experiment | 9/16 pSB1C3(EcoRI+SpeI) | 49.5µg/mL | 9/15 Pcon-RBS-tetR-DT(EcoRI+SpeI) | 59.0µg/mL | xx µg/mL
|
| experiment | 9/16 pSB1C3(EcoRI+SpeI) | 49.5µg/mL | 9/15 Pcon-PT181-atte-DT(EcoRI+SpeI) | 21.0µg/mL | xx µg/mL
|
| experiment | 9/16 pSB1C3(EcoRI+SpeI) | 49.5µg/mL | 9/15 Pcon-apt12-112-DT(EcoRI+SpeI) | 17.0µg/mL | xx µg/mL
|
| Lane | DNA | Enzyme
|
| 1 | 1kbpladder | --
|
| 2 | pSB1C3 | E+S
|
| 3
|
| 4
|
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230
|
| pSB1C3 | 10.0 | 1.84 | 0.40
|
| Lane | DNA | Enzyme
|
| 1 | 1kbpladder | --
|
| 2 | pSB4K5 | X+P
|
| 3
|
| 4
|
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230
|
| pSB4K5 | 7.6 | 1.81 | 0.32
|
| Lane | DNA | Enzyme
|
| 1 | 1kbpladder | --
|
| 2 | Pcon-RBS-tetR-DT | X+P
|
| 3
|
| 4
|
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230
|
| Pcon-RBS-tetR-DT | 8.0 | 2.40 | 1.14
|
| Lane | DNA | Enzyme
|
| 1 | 1kbpladder | --
|
| 2 | pSB1C3 | E+P
|
| 3
|
| 4
|
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230
|
| pSB1C3 | 9.1 | 2.17 | 0.35
|
Mutation PCR
Kinose
| pC KaiABC(100pg) | Prime STAR(2x) | Primer(KaiA-Bsai MuT-Fwd) | Primer(KaiA-Bsai MuT-Rev) | MilliQ | total
|
| 1.0 | 25.0 | 1.0 | 1.0 | 22.0 | 50.0
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 96°C | 98°C | 65°C | 72°C | --
|
| 5m | 10s | 15s | 40s | 30cycles
|
| pC KaiABC(150pg) | Prime STAR(2x) | Primer(KaiA-Bsai MuT-Fwd) | Primer(KaiA-Bsai MuT-Rev) | MilliQ | total
|
| 1.5 | 25.0 | 1.0 | 1.0 | 21.5 | 50.0
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 96°C | 98°C | 65°C | 72°C | --
|
| 5m | 10s | 15s | 40s | 30cycles
|
</div>
| pC KaiABC(200pg) | Prime STAR(2x) | Primer(KaiA-Bsai MuT-Fwd) | Primer(KaiA-Bsai MuT-Rev) | MilliQ | total
|
| 2 | 25.0 | 1.0 | 1.0 | 21.0 | 50.0
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 96°C | 98°C | 65°C | 72°C | --
|
| 5m | 10s | 15s | 40s | 30cycles
|
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