From 2013.igem.org
Sep 2
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Colony PCR
Tatsui
| Sample | base pair
|
| 9/1 RBS-lysis2-DT-(1) | 985
|
| Ptet-RBS-lacZα-DT-(1) | 765
|
| Plac-RBS-lacZα-DT-(1) | 765
|
| Plac-RBS-lacZα-DT-(2) | 765
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 5min | 30s | 30s | 1min | 30cycles
|
Electrophoresis
No name
| Lane | Sample
|
| 1 | 100bp ladder
|
| 2 | 9/1 RBS-lysis2+DT -(1)
|
| 3 | 9/1 Ptet+RBS-lacZα-DT -(1)
|
| 4 | 9/1 Plac+RBS-lacZα-DT -(1)
|
| 5 | 9/1 Plac+RBS-lacZα-DT -(2)
|
| 6 | 100bp ladder
|
Restriction Enzyme Digestion
Nakamoto
| | pSB1C3-(1) | EcoRI | SpeI | XbaI | PstI | BufferB | BufferD | BSA | MilliQ | total
|
| 2 cuts(EcoRI+SpeI) | 7µL | 1µL | 1µL | 0µL | 0µL | 3µL | 0µL | 0.3µL | 17.7µL | 30µL
|
| NC(EcoRI+SpeI) | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0µL | 0.1µL | 8.6µL | 10µL
|
| 2 cuts(XbaI+PstI) | 7µL | 0µL | 0µL | 1µL | 1µL | 0µL | 3µL | 0.3µL | 17.7µL | 30µL
|
| NC(XbaI+PstI) | 0.3µL | 0µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0.1µL | 8.6µL | 10µL
|
| | 8/21 tRMA-spinach(1) | EcoRI | SpeI | BufferB | BSA | MilliQ | total
|
| 2 cuts | 8µL | 1.0µL | 1.0µL | 3µL | 0.3µL | 17.6µL | 30µL
|
| NC | 0.5µL | 0µL | 0µL | 1µL | 0.1µL | 8.4µL | 10µL
|
| | 8/21 tetR aptamer12_1R-(1) | EcoRI | SpeI | BufferB | BSA | MilliQ | total
|
| 2 cuts | 6µL | 1.0µL | 1.0µL | 3µL | 0.3µL | 18.7µL | 30µL
|
| NC | 0.4µL | 0µL | 0µL | 1µL | 0.1µL | 8.5µL | 10µL
|
| | 8/21 pT181 attenuator-(2) | EcoRI | SpeI | XbaI | PstI | BufferB | BufferD | BSA | MilliQ | total
|
| 2 cuts(EcoRI+SpeI) | 8µL | 1µL | 1µL | 0µL | 0µL | 3µL | 0µL | 0.3µL | 16.3µL | 30µL
|
| NC(EcoRI+SpeI) | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0µL | 0.1µL | 8.4µL | 10µL
|
| 2 cuts(XbaI+PstI) | 8µL | 0µL | 0µL | 1µL | 1µL | 0µL | 3µL | 0.3µL | 16.3µL | 30µL
|
| NC(XbaI+PstI) | 0.5µL | 0µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0.1µL | 8.4µL | 10µL
|
Liquid Culture
Kojima
| Sample | medium
|
| tRNA-Spinach-1 | Plusgrow medium(+CP)
|
| tetR aptamaer12_1R-1 | Plusgrow medium(+CP)
|
| pT181 attenuator-1 | Plusgrow medium(+CP)
|
| pT181 antisense-1 | Plusgrow medium(+CP)
|
Master Plate
Kojima
| Number | Use LB plate(+CP)
|
| 1 | tRNA Spinach-1
|
| 2 | tetR aptamaer12_1R
|
| 3 | tetR aptamaer12_P
|
| 4 | tetR aptamaer12_1M
|
| 5 | pT181 attenuator-2
|
| 6 | Fusion1 attenuator-1
|
| 7 | Fusion3m2 attenuator-1
|
| 8 | pT181 antisense-1
|
| 9 | Fusion1 antisense-1
|
| 10 | Fuaion6 antisense-1
|
Electrophoresis
Nakamoto & Tatsui
| Lane | Sample | Enzyme1 | Enzyme2
|
| 1 | 100bp ladder | -- | --
|
| 2 | pSB1C3 -(1) | EcoRI | SpeI
|
| 3 | pSB1C3 -(1) | -- | --
|
| 5 | pSB1C3 -(1) | XbaI | PstI
|
| 6 | pSB1C3 -(1) | -- | --
|
| 7 | pT181 attenuator (2) | EcoRI | SpeI
|
| 8 | pT181 attenuator (2) | -- | --
|
| 9 | pT181 attenuator (2) | XbaI | SpeI
|
| 10 | pT181 attenuator (2) | -- | --
|
| 11 | tetR-aptamer 12_1R | EcoRI | SpeI
|
| 12 | tetR-aptamer 12_1R | -- | --
|
| 13 | tRNA Spinach (1) | EcoRI | SpeI
|
| 14 | 100bp ladder | -- | --
|
| 15 | tRNA Spinach (1) | -- | --
|
No name
| Lane | DNA | Enzyme
|
| 1 | 100bp ladder | -
|
| 2 | pSB1C3(EcoRI+SpeI) | EcoRI&SpeI
|
| 3
|
| 4 | -- | --
|
| 5 | 8/24 tetR aptamer 12_1R | EcoRI&SpeI
|
| 6
|
| Lane | DNA | Enzyme
|
| 1 | 100bp ladder | -
|
| 2 | pT181 attenuator(2)(EcoRI+SpeI) | EcoRI&SpeI
|
| 3
|
| 4 | -- | --
|
| 5 | pT181 attenuator(2)(XbaI+PstI) | EcoRI&SpeI
|
| 6
|
| 7 | -- | --
|
| 8 | pT181 attenuator(2)(XbaI+PstI) | EcoRI&SpeI
|
| 9
|
| 10 | -- | --
|
| 11 | Spinach | EcoRI&SpaI
|
| 12
|
| Name | concentration[µg/mL] | 260/280 | 260/230
|
| pSB1C3 (EcoRI&SpeI) | 3.0 | 2.18 | 0.31
|
| pSB1C3 (XbaI&PstI) | 4.7 | 2.25 | 0.36
|
| pT181 attenuator-(2)(EcoR&SpeI) | 8.6 | 2.74 | 0.01
|
| pT181 attenuator-(2) (XbaI&PstI) | 16.5 | 2.46 | 0.03
|
| Spinach (EcoRI&SpeI) | 2.8 | 2.98 | 0.27
|
| tetR aptamer12_1R (EcoRI&SpeI) | 50.4 | 28.07 | 1.97
|
Colony PCR
No name
| Sample | base pair
|
| 9/1 Spinach(pSB1C3) | about 450
|
| 9/1 Spinach-DT | 596
|
| 9/1 RBS-lysis1-DT | 613
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 5min | 30s | 30s | 40s | 30cycles
|
| Sample | base pair
|
| 9/1 Ptet-RBS-lacZα-DT-2 | 738
|
| 9/1 Ptet-RBS-lacZα-DT-3 | 738
|
| 9/1 Ptet-RBS-lacZα-DT-4 | 738
|
| 9/1 Ptet-RBS-lacZα-DT-5 | 738
|
| 9/1 Ptet-RBS-lacZα-DT-6 | 738
|
| 9/1 RBS-lysis2-DT-2 | 985
|
| 9/1 RBS-lysis2-DT-3 | 985
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 5min | 30s | 30s | 1min | 30cycles
|
Electrophoresis
No name
| Lane | Sample
|
| 1 | 100bp ladder
|
| 2 | Spinach(pSB1C3)
|
| 3 | Spinach-DT
|
| 4 | RBS-lysis1-DT
|
| 5 | 100bp ladder
|
Master Plate
No name
| Number | Use LB plate(+CP)
|
| 1 | 9/1 Spinach(pSB1C3)
|
| 2 | 9/1 Spinach-DT-(1)
|
Liquid Culture
No name
| Sample | medium
|
| pT181 attenuator | Plusgrow medium (+CP)
|
| pT181 antisense | Plusgrow medium (+CP)
|
| Spinach | Plusgrow medium (+CP)
|
- incubated at 37 °C for 1 hour
Transformation
No name
| Name | Sample | Competent Cells | Total | Plate
|
| 8/28 RBS-lysis3-DT | 3µL | 30µL | 33µL | CP
|
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