From 2013.igem.org
Sep 4
Ligation
No name
| state | Vector | Inserter | Ligation High ver.2
|
| experiment | 9/3 DT(EcoRI&XbaI) | 3.2µL | 9/3 RBS-lysis1 | 2.4µL | 2.7µL
|
| experiment | 9/3 DT(EcoRI&XbaI) | 3.0µL | 9/3 RBS-lysis2 | 4.7µL | 4.0µL
|
| experiment | 9/3 DT(EcoRI&XbaI) | 3.0µL | 9/3 RBS-lysis3 | 8.8µL | 4.0µL
|
| experiment | 9/3 DT(EcoRI&XbaI) | 3.0µL | 9/3 aptamer12_1R(EcoRI&SpeI) | 1.9µL | 2.45µL
|
| experiment | 9/2 pSB1C3(XbaI&PstI) | 10.6µL | 9/3 pT181attenuator (XbaI& PstI) | 3.2µL | 4.0µL
|
| experiment | 9/2 pSB1C3(XbaI&PstI) | 9.6µL | 9/3 pT181antisense (XbaI& PstI) | 2.1µL | 4.0µL
|
| experiment | 9/2 pSB1C3(EcoRI&SpeI) | 16.7µL | 9/3 aptamer12_1R(EcoRI&SpeI) | 2.0µL | 4.0µL
|
incubate 16 °C 1 hour
Transformation
Tatsui
| Name | Sample | Competent Cells | Plate
|
| 9/4 RBS-lysis1-DT | 2µL | 20µL | Amp
|
| 9/4 RBS-lysis2-DT | 2µL | 20µL | Amp
|
| 9/4 RBS-lysis3-DT | 2µL | 20µL | Amp
|
| 9/4 aptamer12_1R-DT | 2µL | 20µL | Amp
|
| 9/4 pT181attenuator –pSB1C3 | 2µL | 20µL | Amp
|
| 9/4 pT181antisense-pSB1C3 | 2µL | 20µL | Amp
|
| 9/4 aptamer12_1R –pSB1C3 | 2µL | 20µL | Amp
|
| 9/4 pSB4K5(2013plate5 56) | 2µL | 20µL | Amp
|
| 9/3 Mutagenesis product 1(KaiABC) | 2µL | 20µL | Amp
|
| 9/3 Mutagenesis product 2(NC) | 2µL | 20µL | Amp
|
Miniprep
No name
| DNA | concentration[µg/mL] | 260/280 | 260/230
|
| DT | 193.1 | 1.93 | 1.81
|
| Spinach-DT | 373.3 | 1.92 | 1.69
|
| RBS-lysis1 | 332.0 | 1.95 | 1.62
|
Master Plate
Hirano
| Number | Use LB plate (+CP)
|
| 1 | 8/10 Plac(CP)-2
|
| 2 | 8/10 Plac(CP)-2
|
| 3 | 8/10 Plac(CP)-2
|
| Number | Use LB plate (+Amp)
|
| 1 | 8/9 J23100(Amp)-1
|
| 2 | 8/9 J23100(Amp)-1
|
| 3 | 8/9 J23100(Amp)-1
|
Liquid Culture
Hirano
| Sample | medium
|
| 8/10 Plac(CP)-2 | Plusgrow medium(+CP)
|
| 8/9 J23100(Amp)-1 | Plusgrow medium(+Amp)
|
PCR
Hirano
| 8/29 Pλ-RBS-luxI-DT | KOD plus | 10x buffer | dNTP | MgSO4 | F primer (RBS-luxI-DT-cloning-fwd) | R primer(RBS-luxI-DT-cloning-rev) | MilliQ | total
|
| 0.3µL | 0.5µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 16.2µL | 25µL
|
| 8/31 Pbad/araC-RBS-RFP(1) | KOD plus | 10x buffer | dNTP | MgSO4 | F primer (Pbad/araC-pSB1C3-cloning-fond) | R primer(Pbad/araC-cloning-rex) | MilliQ | total
|
| 0.3µL | 0.5µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 16.2µL | 25µL
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 65°C | 68°C | --
|
| 5min | 10s | 30s | 3min10s | 30cycles
|
Restriction Enzyme Digestion
No name
| | 8/21 Pconst | SpeI | PstI | Buffer | BSA | MilliQ | total
|
| 2 cuts | 10.5µL | 1.0µL | 1.0µL | 3.0µL | 0.3µL | 14.2µL | 30 µL
|
| NC | 0.5µL | 0µL | 0µ | 1.0µL | 0.1µL | 8.4µL | 10 µL
|
| | 8/29 Plac | SpeI | PstI | Buffer | BSA | MilliQ | total
|
| 2 cuts | 13.0µL | 1.0µL | 1.0µL | 3.0µL | 0.3µL | 11.7µL | 30 µL
|
| NC | 0.7µL | 0µL | 0µ | 1.0µL | 0.1µL | 8.2µL | 10 µL
|
| | 8/20 Ptet | SpeI | PstI | Buffer | BSA | MilliQ | total
|
| 2 cuts | 9.0µL | 1.0µL | 1.0µL | 3.0µL | 0.3µL | 15.7µL | 30 µL
|
| NC | 0.5µL | 0µL | 0µ | 1.0µL | 0.1µL | 8.4µL | 10 µL
|
| | 8/20 RBS-tetR-DT | XbaI | PstI | Buffer | BSA | MilliQ | total
|
| 2 cuts | 8.5µL | 1.0µL | 1.0µL | 3.0µL | 0.3µL | 16.2µL | 30 µL
|
| NC | 0.5µL | 0µL | 0µ | 1.0µL | 0.1µL | 8.4µL | 10 µL
|
| | spinach -DT | XbaI | PstI | Buffer | BSA | MilliQ | total
|
| 2 cuts | 5.4µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 16.6µL | 30 µL
|
| NC | 0.3µL | 0µL | 0µ | 1.0µL | 1.0µL | 7.7µL | 10 µL
|
| | 8/17 RBS-lacZα-DT | XbaI | PstI | Buffer | BSA | MilliQ | total
|
| 2 cuts | 11µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 11µL | 30 µL
|
| NC | 0.5µL | 0µL | 0µ | 1.0µL | 1.0µL | 7.5µL | 10 µL
|
Transformation
No name
| Name | Sample | Competent Cells | Total | Plate
|
| Ptrc KaiC | 1µL | 10µL | 11µL | Amp
|
| Ptrc KaiB | 1µL | 10µL | 11µL | Amp
|
| pSΩ | 1µL | 10µL | 11µL | Amp
|
Electrophoresis
No name
1
| Lane | Sample | Enzyme1 | Enzyme2
|
| 1 | 100bp ladder | -- | --
|
| 2 | Pconst | SpeI | PstI
|
| 3 | Pconst NC | -- | --
|
| 4 | Plac | SpeI | PstI
|
| 5 | Plac NC | -- | --
|
| 6 | 100bp ladder | -- | --
|
File:Igku xxxxxx.xxx
2
| Lane | Sample | Enzyme1 | Enzyme2
|
| 1 | 100bp ladder | -- | --
|
| 2 | Ptet | SpeI | PstI
|
| 3 | Ptet NC | -- | --
|
| 4 | RBS-tetR-DT | XbaI | PstI
|
| 5 | RBS-tetR-DT NC | -- | --
|
| 6 | 100bp ladder | -- | --
|
File:Igku xxxxxx.xxx
No name
1
| Lane | DNA | Enzyme
|
| 1 | 100bp ladder | --
|
| 2 | Pcon | SpeI&PstI
|
| 3 | Pcon | SpeI&PstI
|
| 4 | -- | --
|
| 5 | Plac | SpeI&PstI
|
| 6 | Plac | SpeI&PstI
|
2
| Lane | DNA | Enzyme
|
| 1 | 100bp ladder | --
|
| 2 | Ptet | SpeI&PstI
|
| 3 | Ptet | SpeI&PstI
|
| 4 | -- | --
|
| 5 | RBS-tetR-DT | XbaI&PstI
|
| 6 | RBS-tetR-DT | XbaI&PstI
|
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | quantity | concentration[µg/mL] | 260/280 | 260/230
|
| Pcon | -- | 13.2 | 1.99 | -1.64
|
| Plac | -- | 15.3 | 2.07 | -0.39
|
| Ptet | -- | 30.1 | 1.97 | -0.97
|
| RBS-tetR-DT | -- | 3.0 | 2.44 | 0.88
|
No name
| Lane | DNA | Enzyme
|
| 1 | 100bp ladder | --
|
| 2 | spinach-DT | XbaI&PstI
|
| 3 | spinach-DT | XbaI&PstI
|
| 4 | -- | --
|
| 5 | RBS-lacZα-DT | XbaI&PstI
|
| 6 | RBS-lacZα-DT | XbaI&PstI
|
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | quantity | concentration[µg/mL] | 260/280 | 260/230
|
| spinach-DT | -- | 2.1 | 2.10 | -0.05
|
| RBS-lacZα-DT | -- | 3.3 | 2.59 | -0.07
|
Master Plate
No name
| Number | Use LB plate (+Amp)
|
| 4 | 8/9 BBa_J23100-3
|
| 5 | 8/9 BBa_J23100-4
|
| 6 | 8/9 BBa_J23100-5
|
| Number | Use LB plate
|
| 1 | JM109-1
|
| 2 | JM109-2
|
| 3 | JM109-3
|
| 4 | JM109-4
|
Liquid Culture
Hirano
| Sample | medium
|
| 8/9 BBa_J23100-3 | Plusgrow medium(+Amp)
|
| JM109-1(Master plate) | LB medium
|
Colony PCR
Hirano
| Sample | base pair
|
| 8/9 BBa_J23100 3 | --
|
| 8/9 BBa_J23100 4 | --
|
| 8/9 BBa_J23100 5 | --
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 5min | 30s | 30s | 30s | 30cycles
|
"
