From 2013.igem.org
Sep 17
Liquid Culture
Tatsui and Hirano
| Sample | medium
|
| Spinach-DT | LB medium(+CP)
|
| Pcon-Spinach-DT | LB medium(+Amp)
|
| Pcon-pT181 antisense-Spinach-DT | LB medium(+Amp)
|
PCR
Hirano
| Pcon-tetR aptamer-DT(VF) | BigDye | 5x buffer | primer(VF) | MilliQ | total
|
| 1.3 | 0.5 | 1.75 | 0.5 | 0.45 | 10.5
|
| Pcon-tetR aptamer-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 | --
|
| 2 min | 10 sec | 5 sec | 4 min | 30 cycles
|
Electrophoresis
No name
| Lane | Sample | Enzyme1 | Enzyme2
|
| 1 | 1kbp ladder | -- | --
|
| 2 | Pcon-RBS-tetR-DT | XbaI | PstI
|
| 3 | Pcon-RBS-tetR-DT | -- | --
|
| 4 | pSB4K5 | XbaI | PstI
|
| 5 | pSB4K5 | -- | --
|
| 6 | pSB1C3 | EcoRI | SpeI
|
| 7 | pSB1C3 | EcoRI | PstI
|
| 8 | pSB1C3 | -- | --
|
| 9 | pT181 attenuator | EcoRI | SpeI
|
| 10 | pT181 attenuator | -- | --
|
| 11 | 1kbp ladder | -- | --
|
Ligation
No Name
| state | Vector | Inserter | Ligation High ver.2
|
| experiment | 8/28 Plux (SpeI & PstI) | 5.0 µL | 9/13 RBS-lysis1-DT (XbaI & PstI) | 3.5 µL | 3.5 µL
|
| experiment | 8/28 Plux (SpeI & PstI) | 5.0 µL | 9/13 RBS-lysis2-DT (XbaI & PstI) | 3.6 µL | 3.5 µL
|
| experiment | 8/28 Plux (SpeI & PstI) | 11.0 µL | 9/13 RBS-lysis3-DT (XbaI & PstI) | 9.5 µL | 3.5 µL
|
| experiment | 9/14 Plac (SpeI & PstI) | 2.4 µL | 9/16 pT181 attenuator-DT (XbaI & PstI) | 4.8 µL | 3.5 µL
|
| experiment | 9/14 Plac (SpeI & PstI) | 2.4 µL | 9/3 pT181 antisense (XbaI & PstI) | 2.0 µL | 2.2 µL
|
| experiment | 9/14 Plac (SpeI & PstI) | 2.4 µL | 9/10 aptamer 12_1R-DT (XbaI & PstI) | 1.8 µL | 2.1 µL
|
| experiment | 9/13 pSB1C3 (XbaI & PstI) | 2.0 µL | 9/15 Pcon-PT181 antisense-Spinach-DT (XbaI & PstI) | 12.0 µL | 3.5 µL
|
| experiment | 9/16 pSB1C3 (EcoRI & SpeI) | 1.0 µL | 9/15 Pcon-Spinach-DT (EcoRI & SpeI) | 4.1 µL | 2.5 µL
|
| experiment | 9/16 pSB1C3 (EcoRI & SpeI) | 1.0 µL | 9/15 Pcon-RBS-tetR-DT (EcoRI & SpeI) | 2.0 µL | 1.5 µL
|
| experiment | 9/16 pSB1C3 (EcoRI & SpeI) | 1.0 µL | 9/15 Pcon-PT181 attenuator-DT (EcoRI & SpeI) | 2.4 µL | 1.7 µL
|
| experiment | 9/16 pSB1C3 (EcoRI & SpeI) | 1.0 µL | 9/15 Pcon-aptamer 12_1R-DT (EcoRI & SpeI) | 2.0 µL | 1.5 µL
|
| experiment | 9/13 pSB1C3 (XbaI & PstI) | 2.0 µL | 9/16 Pcon-pT181-attenuator (XbaI & PstI) | 4.4 µL | 3.2 µL
|
Mutation PCR
Kinose
| pC KaiABC(100pg/µL) | 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
|
| 1.5 | 25.0 | 1.0 | 1.0 | 21.5 | 50.0
|
| 2.0 | 25.0 | 1.0 | 1.0 | 21.0 | 50.0
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 96 °C | 98 °C | 65 °C | 72 °C | --
|
| 5 min | 10 s | 15 s | 40 s | 30 cycles
|
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