From 2013.igem.org
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| ===Transformation=== | | ===Transformation=== |
Revision as of 12:17, 26 September 2013
Sep 22
Colony PCR
Tatsui
Sample | base pair
|
9/20 Plac-PT181 attenuator(1~4) | 664
|
9/20 Plac-PT181 antisense(1~6) | 369
|
9/20 Pcon-PT181 attenuator-aptamer-DT(1~6) | 859
|
9/20 Pcon-PT181 antisense-spinach-DT(1~8) | 723
|
Electrophoresis
Nakamoto
Lane | Sample
|
1 | Pcon-apt12_1R-DT(E-1A)
|
2 | Pcon-spinach-DT(E-1A)
|
3 | Pcon-pT181 attenuator(E-1A)
|
4 | DT(1-3)
|
5 | tetR aptamer 12_1R-DT(1-3)
|
6 | 100bp ladder
|
7 | Pcon-pT181 attenuator(1-SA)
|
8 | spinach-DT(2-1)
|
9 | Pcon-pT181 attenuator(E-0)
|
10 | Pcon-pT181 attenuator(2-SA)
|
11 | spinach-DT(3-2)
|
12 | Ptet-PT181 anisense
|
Miniprep
Honda
DNA | concentration[µg/mL] | 260/280 | 260/230
|
9/21 spinach-DT | 197.9 | 1.67 | 1.54
|
9/21 Plux | 125.9 | 1.71 | 1.27
|
9/21 Plux-RBS-GFP-DT-(1) | 168.0 | 1.62 | 1.73
|
9/21 Pcon-PT181 attenuator-(1) | 368.3 | 1.57 | 1.62
|
9/21 RBS-GFP-DT | 532.8 | 1.75 | 1.78
|
9/21 Pcon-RBS-GFP-DT | 845.2 | 2.02 | 1.70
|
Colony PCR
Honda
Sample | base pair
|
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1~2) | 283
|
PreDenature | Denature | Annealing | Extension | cycle
|
94°C | 94°C | 55°C | 68°C | --
|
5min | 30s | 30s | 2min10s | 30cycles
|
No name
Lane | DNA | Enzyme
|
1 | 100bp ladder | --
|
2~4 | Pcon-aptamer12-1R-DT(E-1A) | --
|
6~8 | Pcon-spinach-DT(E-1A) | --
|
10~12 | Pcon-attenuator PT181(E-1A) | --
|
Lane | DNA | Enzyme
|
1 | 100bp ladder | --
|
2~4 | DT(1-3) | --
|
6~8 | aptamer12-1RDT(1-3) | --
|
10~12 | Pcon-PT181 attenuator(1-5A) | --
|
Name | concentration[µg/mL] | 260/280 | 260/230
|
Pcon-aptamer12-1R-DT(E-1A) | 31.2 | 1.87 | 0.30
|
Pcon-spinach-DT(E-1A) | 25.6 | 1.88 | 0.99
|
Pcon-attenuator PT181(E-1A) | 25.3 | 1.99 | 0.86
|
DT(1-3) | 22.1 | 1.82 | 0.20
|
aptamer12-1RDT(1-3) | 33.5 | 1.99 | 0.05
|
Pcon-PT181 attenuator(1-5A) | 36.9 | 1.12 | 0.96
|
digestion
No name
| 9/16 Plac | XbaI | SpeI | PstI | BufferB | BSA | MilliQ | total
|
2cuts | 12.5µL | 0µL | 1µL | 1µL | 3µL | 3µL | 9.5µL | 30µL
|
NC | 0.8µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.4µL | 10µL
|
| 9/22 Plux | XbaI | SpeI | PstI | BufferB | BSA | MilliQ | total
|
2cuts | 16µL | 0µL | 1µL | 1µL | 3µL | 3µL | 14µL | 30µL
|
NC | 0.8µL | 0µL | | 0µL | 0µL | 1µL | 1µL | 7.2µL | 10µL
|
| 9/20 RBS-tetR-2R-DT | XbaI | SpeI | PstI | BufferD | BSA | MilliQ | total
|
2cuts | 8.5µL | 1µL | 0µL | 1µL | 3µL | 3µL | 13.5µL | 30µL
|
NC | 0.4µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL
|
No name
Lane | DNA | Enzyme
|
1 | 100bp ladder | --
|
2~4 | Pcon-attenuator PT181(E-0) | --
|
Lane | DNA | Enzyme
|
1 | 100bp ladder | --
|
2~4 | Pcon-attenuator PT181(E-0) | --
|
Lane | DNA | Enzyme
|
2 | 100bp ladder | --
|
4~6 | Pcon-attenuator PT181(2-SA) | --
|
Lane | DNA | Enzyme
|
1 | 100bp ladder | --
|
3~5 | spinach-DT(3-2) | --
|
8~10 | Ptet-antisense PT181(1-3) | --
|
Name | concentration[µg/mL] | 260/280 | 260/230
|
Pcon-attenuator PT181(E-0) | 2.4 | 1.80 | 0.17
|
Pcon-attenuator PT181(2-SA) | 10.8 | 1.85 | 0.54
|
spinach-DT(3-2) | 36.7 | 1.80 | 0.75
|
Ptet-antisense PT181(1-3) | 16.8 | 1.74 | 0.78
|
Colony PCR
No name
Sample | base pair
|
9/20 Pcon-PT181 attenuator-aptamer12-1R-DT-(7~14) | 859
|
Colony PCR
Tatsui
Sample | base pair
|
PconPT181antisense-spinach-DT(9~15) | 723
|
Ligation
No name
state | Vector | Inserter | Ligation High ver.2
|
experiment | 9/13 Pcon-PT181 attenuator (SpeI & PstI) | 1.7 µL | 9/18 aptamer-1R-DT (XbaI & PstI) | 5.4µL | 3.5µL
|
experiment | 9/12 Plac(SpeI & PstI) | 2.4 µL | 9/18 aptamer-1R-DT (XbaI & PstI) | 6.1µL | 4.5 µL
|
experiment | 9/13 Ptet(SpeI & PstI) | 1.7 µL | 9/10 Plux-RBS-GFP-DT | 7.0 µL | 4.4 µL
|
experiment | 9/21 RBS-GFP-DT (EcoRI & XbaI) | 0.6 µL | 9/18 aptamer 12-1R-DT (XbaI & PstI) | 5.6µL | 3.1 µL
|
experiment | 9/13 PSB1C3(XbaI & PstI) | 1.9 µL | 9/16 PT181 attenuator(XbaI & PstI) | 4.6µL | 3.3 µL
|
Liquid Culture
No name
Sample | medium
|
9/20 Plac-antisense-1 | --
|
9/20 Plac-attenuator-1 | --
|
9/20 Plac-antisense-1 | --
|
9/20 Plac-attenuator-1 | --
|
digestion
No name
| 9/8 Pcon-PT181 antisense | EcoRI | SpeI | BufferM | MilliQ | total
|
2cuts | 8.3µL | 1µL | 1µL | 3µL | 16.7µL | 30µL
|
NC | 0.4µL | 0µL | 0µL | 1µL | 8.3µL | 10µL
|
| 9/7 Pcon-RBS-tetR-DT | EcoRI | XbaI | SpeI | PstI | BufferD | BSA | MilliQ | total
|
2cut | 14µL | 1µL | 1µL | 0µL | 0µL | 3µL | 18.2µL | 30µL
|
NC | 0.7µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.3µL | 10µL
|
| 9/22 RBS-GFP-DT | EcoRI | XbaI | SpeI | PstI | BufferD | BSA | MilliQ | total
|
2cuts | 3.8µL | 1µL | 1µL | 0µL | 0µL | 3µL | 3µL | 18.2µL | 30µL
|
NC | 0.2µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.8µL | 10µL
|
| 9/6 aptamer12-1R-DT | EcoRI | XbaI | SpeI | PstI | BufferD | BSA | MilliQ
|
2cut | 8µL | 1µL | 1µL | 0µL | 0µL | 3µL | 3µL | 16µL
|
NC | 0.4µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.6µL
|
incubate 37°C 1hour
No name
Lane | DNA | Enzyme
|
1 | 1kbp ladder | --
|
3~5 | Pcon-PT181 antisense (EcoRI&SpaI) | --
|
Transformation
No name
Name | Sample | Competent Cells | Plate
|
9/20 Plac-aptamer12-1R-DT(XbaI&PstI) | 2 µL | 20 µL | Amp
|
9/20 pT181 attenuator(1C3)(EcoRI&SpeI) | 2 µL | 20 µL | CP
|
9/17 pT181 attenuator(1C3)(XbaI&PstI) | 2 µL | 20 µL | CP
|
9/20 Pcon-pT181 attenuator(SpaI&PstI)+aptamer12-1R-DT(XbaI&PstI) | 2 µL | 20 µL | Amp
|
kaiABC | 2 µL | 20 µL | Amp
|
9/22 pSB1C3-RpaB(XbaI&PstI) | 2 µL | 20 µL | Amp
|
9/22 pSB1C3-pT181 attenuator(XbaI&PstI) | 2 µL | 20 µL | Amp
|
9/22 Ptet(SpaI&PstI)+RBS-GFP-DT(XbaI&PstI) | 2 µL | 20 µL | CP
|
9/22 RBS-GFP-DT+Pcon-attenuator pT181 (EcoRI&SpeI) | 2 µL | 20 µL | CP
|
9/22 pT181 attenuator(XbaI&PstI)(1C3) | 2 µL | 20 µL | CP
|
PCR
No name
Sample | base pair
|
Pcon-RBS-tetR-DT(1-2Ⓐ) | --
|
Pcon-RBS-tetR-DT(3-2Ⓐ) | --
|
Pcon-RBS-GFP-DT(1-SⒶ) | --
|
Pcon-RBS-GFP-DT(2-SⒶ) | --
|
Pcon-tetR-DT(E-2Ⓐ) | --
|
PreDenature | Denature | Annealing | Extension | cycle
|
94 °C | 94 °C | 57 °C | 68 °C | --
|
2 min | 10 s | 30 s | 1min5s | 30 cycles
|
Electrophoresis
No name
Lane | Sample
|
1 | 1kbp ladder
|
2 | Pcon-RBS-tetR-DT(1-2Ⓐ)
|
3 | Pcon-RBS-tetR-DT(3-2Ⓐ)
|
4 | Pcon-RBS-GFP-DT(1-SⒶ)
|
5 | Pcon-RBS-GFP-DT(2-SⒶ)
|
6 | Pcon-tetR-DT(E-2Ⓐ)
|