From 2013.igem.org
Aug 28
Electrophoresis
No name
| Lane | Sample
|
| 1 | 100bp
|
| 2 | SasA
|
| 3 | RpaA
|
| 4 | RpaB
|
| 5 | PkaiBC
|
| 6 | PCR NC
|
| 7 | 100bp
|
Miniprep
No name
| DNA | concentration[µg/mL] | 260/280 | 260/230
|
| 8/27 Plux | 173.8 | 1.95 | 1.45
|
Ligation
No name
| state | Vector | Inserter
|
| experiment | 8/17 DT-1 | 3.0 | 8.0
|
- Samples were evaporeted used evaporator into about 3 µL.
| sample | MilliQ | Ligation High | total
|
| 3 | 4 | 3.5 | 10.5
|
- incubate one hour at 16 °C
No name
| Lane | DNA
|
| 1 | 100bpladder
|
| 3 | SasA(8/27 Genome PCR production)
|
| 5 | RpaA(8/27 Genome PCR production)
|
| 7 | RpaB(8/27 Genome PCR production)
|
| 9 | PkaiBC(8/27 Genome PCR production)
|
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230
|
| RpaA | 15.4 | 1.88 | 0.03
|
| SasA | 21.6 | 1.66 | 0.79
|
| PkaiBC | 16.3 | 1.79 | 0.68
|
| RpaB | 26.7 | 1.77 | 0.82
|
Colony PCR
No name
| Sample | base pair
|
| 8/21 RBS-lysis2-(3) | 772
|
| 8/21 RBS-lysis2-(4) | 772
|
| 8/21 RBS-lysis2-(5) | 772
|
| 8/21 RBS-lysis2-(6) | 772
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 5min | 30s | 30s | 48s | 30cycles
|
File:Igku Aug19electrophoresis1.png
Restriction Enzyme Digestion
No name
| | 8/22 RBS-lysis1-(1) | EcoRI | SpeI | BufferB | BSA | MilliQ | total
|
| 2 cuts | 9µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 16.7µL | 30µL
|
| NC | 1µL | 0µL | 0µL | 1µL | 0.1µL | 7.9µL | 10µL
|
| | 8/17 RBS-GFP-DT-(2) | XbaI | PstI | BufferD | BSA | MilliQ | total
|
| 2 cuts | 4µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 21.7µL | 30µL
|
| NC | 0.5µL | 0µL | 0µL | 1µL | 0.1µL | 8.4µL | 10µL
|
| | 8/20 Pcon-RBS-GFP-DT-(1) | EcoRI | SpeI | BufferB | BSA | MilliQ | total
|
| 2 cuts | 3µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 22.7µL | 30µL
|
| NC | 0.3µL | 0µL | 0µL | 1µL | 0.1µL | 8.6µL | 10µL
|
| | 8/20 Pcon-RBS-luxR-DT-(1) | EcoRI | XbaI | BufferD | BSA | MilliQ | total
|
| 2 cuts | 2µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 23.7µL | 30µL
|
| NC | 0.3µL | 0µL | 0µL | 1µL | 0.1µL | 8.6µL | 10µL
|
No name
| | 8/28 Plux | SpeI | PstI | BufferD | BSA | MilliQ | total
|
| 2 cuts | 6µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 19.7µL | 30µL
|
| NC | 1µL | 0µL | 0µL | 1µL | 0.1µL | 7.9µL | 10µL
|
| | 8/20 RBS-tetR-DT-(1) | XbaI | PstI | BufferD | BSA | MilliQ | total
|
| 2 cuts | 5µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 20.7µL | 30µL
|
| NC | 0.5µL | 0µL | 0µL | 1µL | 0.1µL | 8.4µL | 10µL
|
Electrophoresis
No name
| Lane | Sample | Enzyme1 | Enzyme2
|
| 1 | 8/22 RBS-lysis1-(1) | EcoRI | SpeI
|
| 2 | 8/22 RBS-lysis1-(1) | -- | --
|
| 3 | 8/17 RBS-GFP-DT-(2) | XbaI | PstI
|
| 4 | 8/17 RBS-GFP-DT-(2) | -- | --
|
| 5 | 8/20 Pcon-RBS-GFP-DT-(1) | EcoRI | XbaI
|
| 6 | 8/20 Pcon-RBS-GFP-DT-(1) | -- | --
|
| 7 | 100bp ladder | -- | --
|
| 8 | 8/20 Pcon-RBS-luxR-DT-(1) | EcoRI | XbaI
|
| 9 | 8/20 Pcon-RBS-luxR-DT-(1) | -- | --
|
| 10 | 8/28 Plux | SpeI | PstI
|
| 11 | 8/28 Plux | -- | --
|
| 12 | 8/20 RBS-tetR-DT | XbaI | PstI
|
| 13 | 8/20 RBS-tetR-DT | -- | --
|
File:Igku Aug28electrophoresis1
No name
| Lane | DNA | Enzyme
|
| 1 | 100bp ladder | --
|
| 2 | 8/20 RBS-lysis1-(1) | EcoRI & SpeI
|
| 3
|
| 4 | -- | --
|
| 5 | 8/28 RBS-GFP-DT-(2) | XbaI & PstI
|
| 6
|
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Lane | DNA | Enzyme
|
| 1 | 100bp ladder | --
|
| 2 | 8/28 Plux | SpeI & PstI
|
| 3
|
| 4 | -- | --
|
| 5 | 8/20 RBS-tetR-DT-(1) | XbaI & PstI
|
| 6
|
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230
|
| Pcon-RBS-GFP-DT (EcoRI&SpeI) | 7.1 | 1.88 | 0.07
|
| Pcon-RBS-luxR-DT (EcoRI&XbaI) | 15.3 | 1.93 | 0.70
|
| Plux (SpeI&PstI) | 4.5 | 1.79 | 0.31
|
| RBS-GFP-DT (XbaI&PstI) | 6.0 | 2.16 | 0.03
|
| RBS-tetR-DT (XbaI&PstI) | 7.6 | 1.91 | 0.38
|
Colony PCR
No name
| Sample | base pair
|
| 8/27 pT181 attenuator-DT-1 | 738
|
| 8/27 pT181 attenuator-DT-2 | 738
|
| 8/27 pT181 attenuator-DT-3 | 738
|
| 8/27 TetR-aptamer 12_1R-DT-1 | 521
|
| 8/27 TetR-aptamer 12_1R-DT-2 | 521
|
| 8/27 pT181 antisense-DT-1 | 542
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 5min | 30s | 30s | 30s | 30cycles
|
File:Igku Aug28electrophoresis3?.png
| Sample | base pair
|
| 8/27 Pλ-luxI-1 | ???
|
| 8/27 Pλ-luxI | ???
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 5min | 30s | 30s | 30min | 3cycles
|
File:Igku Aug19electrophoresis1.png
Colony PCR
No name
| Sample | base pair
|
| 8/21 RBS-lysis2-9 | 772
|
| 8/21 RBS-lysis2-10 | 772
|
| 8/21 RBS-lysis2-11 | 772
|
| 8/21 RBS-lysis2-12 | 772
|
| 8/21 RBS-lysis2-13 | 772
|
| 8/21 RBS-lysis2-14 | 772
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 5min | 30s | 30s | 48s | 30cycles
|
File:Igku Aug28electrophoresis4?.png
Liquid Culture
No name
| Sample | medium
|
| 8/16 Master Plate(CP)-6 DT | Plusgrow medium(+CP)
|
| J23100 Master Plate-1 | Plusgrow medium(+Amp)
|
| Plac Master Plate-1 | Plusgrow medium(+CP)
|
Ligation
No name
| state | Vector | Inserter
|
| experiment | 8/27 DT (EcoRI & XbaI) | 2.9 | 8/28 RBS-lysis1 (EcoRI & SpeI) | 9.5
|
| experiment | 8/28 Plux (SpeI & PstI) | 2.3 | 8/28 RBS-GFP-DT (XbaI & PstI) | 18
|
| experiment | 8/28 Pcon-RBS-luxR-DT (EcoRI & XbaI) | 3.3 | 8/28 Pcon^RBS-GFP-DT-(1) (EcoRI & SpeI) | 28
|
| experiment | 8/27 DT (EcoRI & XbaI) | 2.7 | 8/24 Spinach (EcoRI & SpeI) | 2.4
|
- Samples were evaporeted used evaporator into about 1 µL.
| sample | MilliQ | Ligation High | total
|
| 1 | 3 | 4 | 9
|
- incubate overnight at 4 °C
Liquid Culture
No name
| Sample | medium
|
| 8/26 Pλ-RBS-luxI-DT-1 | Plusgrow medium (+Amp)
|
| 8/26 Pλ-RBS-luxI-DT-2 | Plusgrow medium (+Amp)
|
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