Template:Kyoto/Notebook/Sep 14
From 2013.igem.org
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|experiment||9/13 Pcon-PT181attenuator(SpeI&PstI)||1.7||9/13 RBS-lacZα-DT (XbaI & PstI)||12.5||5 | |experiment||9/13 Pcon-PT181attenuator(SpeI&PstI)||1.7||9/13 RBS-lacZα-DT (XbaI & PstI)||12.5||5 | ||
|- | |- | ||
- | |experiment||9/13 pSB2C3 (XbaI & PstI)||1.9||9/14 RpaB (XbaI & PstI)||16.3|| | + | |experiment||9/13 pSB2C3 (XbaI & PstI)||1.9||9/14 RpaB (XbaI & PstI)||16.3||10.5 |
|- | |- | ||
- | |experiment||9/13 pSB2C3 (XbaI & PstI)||4.7||9/14 PkaiBC (EcoRI & SpeI)||6.7|| | + | |experiment||9/13 pSB2C3 (XbaI & PstI)||4.7||9/14 PkaiBC (EcoRI & SpeI)||6.7||5.7 |
|- | |- | ||
- | |experiment||9/14 RBS-GFP-DT (PstI&XbaI)||0.7||9/14 PkaiBC (EcoRI & SpeI)||2.6|| | + | |experiment||9/14 RBS-GFP-DT (PstI&XbaI)||0.7||9/14 PkaiBC (EcoRI & SpeI)||2.6||1.6 |
|- | |- | ||
- | |experiment||9/14 Plac (SpeI&PstI)||2.4||9/14 RBS-lysis1-DT (XbaI & PstI)||4.9|| | + | |experiment||9/14 Plac (SpeI&PstI)||2.4||9/14 RBS-lysis1-DT (XbaI & PstI)||4.9||3.6 |
|- | |- | ||
- | |experiment||9/14 Plac (SpeI&PstI)||2.4||9/14 RBS-lysis2-DT (XbaI & PstI)||7.0|| | + | |experiment||9/14 Plac (SpeI&PstI)||2.4||9/14 RBS-lysis2-DT (XbaI & PstI)||7.0||4.7 |
|- | |- | ||
- | |experiment||9/14 Plac (SpeI&PstI)||2.4||9/14 RBS-lysis3-DT (XbaI & PstI)||9.2|| | + | |experiment||9/14 Plac (SpeI&PstI)||2.4||9/14 RBS-lysis3-DT (XbaI & PstI)||9.2||5.8 |
|} | |} | ||
Revision as of 09:22, 26 September 2013
Contents |
Sep 14
Restriction Enzyme Digestion
9/14 pSB4K5S | EcoRI | XbaI | SpeI | PstI | BufferB | BufferD | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|---|
2cuts | 10µL | 1µL | 0µL | 1µL | 0µL | 3µL | 0µL | 3µL | 12µL | 30µL |
NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0µL | 1µL | 7.5µL | 10µL |
9/13 Plac | XbaI | PstI | BufferH | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|
1 cut | 9µL | 0µL | 1µL | 3µL | 3µL | 14µL | 30µL |
NC | 1µL | 0µL | 0µL | 1µL | 1µL | 7µL | 10µL |
9/14 PKaiBC | EcoRI | XbaI | SpeI | PstI | BufferB | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2cuts | 11µL | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 11µL | 30µL |
NC | 4µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 4µL | 10µL |
9/14 RpaB | EcoRI | XbaI | SpeI | PstI | BufferD | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2cuts | 13µL | 0µL | 1µL | 0µL | 1µL | 3µL | 3µL | 9µL | 30µL |
NC | 2µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 5µL | 10µL |
8/17 RBS-GFP-DT | EcoRI | XbaI | SpeI | PstI | BufferD | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
1cut | 9µL | 0µL | 1µL | 0µL | 0µL | 3µL | 3µL | 14µL | 30µL |
NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |
- incubated 37°C 1hour
Liquid Culture
Sample | medium |
---|---|
9/11 Plac(BBa-R0011)-(3) | Plusgrow medium(+Amp) |
PCR
genome DNA | MilliQ | Big Dye | 5x buffer | temp(400mg) | primer | total | |
---|---|---|---|---|---|---|---|
Pcon-RBS-luxR-DT | 0.5 | 1.75 | 1.75 | 0.4 | 0.5 | 5.1 | 10.5 |
Pcon-RBS-luxR-DT | 0.5 | 1.75 | 1.75 | 0.4 | 0.5 | 5.1 | 10.5 |
Pcon-RBS-tetR-DT | 0.5 | 1.75 | 1.75 | 3 | 0.5 | 3 | 10.5 |
Pcon-RBS-tetR-DT | 0.5 | 1.75 | 1.75 | 3 | 0.5 | 3 | 10.5 |
Pcon-RBS-luxR-DT | 0.5 | 1.75 | 1.75 | 2.4 | 0.5 | 3.6 | 10.5 |
Plux-RBS-GFP-DT | 0.5 | 1.75 | 1.75 | 1.9 | 0.5 | 4.1 | 10.5 |
Plux-RBS-GFP-DT | 0.5 | 1.75 | 0.5 | 1.9 | 0.5 | 4.1 | 10.5 |
Pcon-RBS-GFP-DT | 0.5 | 1.75 | 0.5 | 1.2 | 0.5 | 4.8 | 10.5 |
RBS-lysis3-DT | 0.5 | 1.75 | 0.5 | 1.0 | 0.5 | 5.0 | 10.5 |
Pcon-RBS-GFP-DT | 0.5 | 1.75 | 0.5 | 1.4 | 0.5 | 4.6 | 10.5 |
Pcon-RBS-GFP-DT | 0.5 | 1.75 | 0.5 | 1.4 | 0.5 | 4.6 | 10.5 |
RBS-lysis3-DT | 0.5 | 1.75 | 1.75 | 0.4 | 0.5 | 5.1 | 10.5 |
Ptet-RBS-spinach-DT | 0.5 | 1.75 | 0.5 | 0.59 | 0.5 | 0.1 | 10.5 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
96°C | 96°C | 50°C | 60°C | -- |
2min | 10s | 5s | 4min | 30cycle |
Restriction Enzyme Digestion
9/14 Plac(1A2) | EcoRI | XbaI | SpeI | PstI | BufferB | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2cuts | 23µL | 0µL | 0µL | 0.3µL | 0µL | 3µL | 3µL | 0µL | 30µL |
9/14 RBS-GFP-DT | EcoRI | XbaI | SpeI | PstI | BufferH | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 23µL | 1µL | 0µL | 0µL | 0µL | 3µL | 3µL | 0µL | 30µL |
Ligation
state | Vector | Inserter | Ligation High ver.2 | ||
---|---|---|---|---|---|
experiment | 9/13 Pcon-PT181attenuator(SpeI&PstI) | 1.7 | 9/13 RBS-lacZα-DT (XbaI & PstI) | 12.5 | 5 |
experiment | 9/13 pSB2C3 (XbaI & PstI) | 1.9 | 9/14 RpaB (XbaI & PstI) | 16.3 | 10.5 |
experiment | 9/13 pSB2C3 (XbaI & PstI) | 4.7 | 9/14 PkaiBC (EcoRI & SpeI) | 6.7 | 5.7 |
experiment | 9/14 RBS-GFP-DT (PstI&XbaI) | 0.7 | 9/14 PkaiBC (EcoRI & SpeI) | 2.6 | 1.6 |
experiment | 9/14 Plac (SpeI&PstI) | 2.4 | 9/14 RBS-lysis1-DT (XbaI & PstI) | 4.9 | 3.6 |
experiment | 9/14 Plac (SpeI&PstI) | 2.4 | 9/14 RBS-lysis2-DT (XbaI & PstI) | 7.0 | 4.7 |
experiment | 9/14 Plac (SpeI&PstI) | 2.4 | 9/14 RBS-lysis3-DT (XbaI & PstI) | 9.2 | 5.8 |
Samples were evaporeted used evaporator into about 7 µL
Colony PCR
Sample | base pair |
---|---|
9/13 Plux+RBS-lysis1-DT (1)~(3) | -- |
9/13 Plux+RBS-lysis2-DT (1)~(3) | - |
9/13 Plux+RBS-lysis3-DT (1)~(2) | - |
Sample | base pair |
---|---|
9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT (1)~(4) | -- |
9/13 Pcon-PT181attenuator+aptamer121R-DT (1)~(2) | - |
9/13 Plac+PT181attenuator (1) | - |
Sample | base pair |
---|---|
Dλ-lux(1) (1)~(2) | -- |
NC | -- |
Liquid Culture
Sample | medium |
---|---|
9/13 Pλ-luxI(1)-1 | Plusgrow medium(+Amp) |
Transformation
Name | Sample | Competent Cells | Total | Plate |
---|---|---|---|---|
9/13 RBS-lysisZα-DT(XbaI&PstI)+9/13 Pcon-PT181 attenuator(SpeI&PstI) | 2µL | 20µL | 22µL | Amp |
9/13 SB2C3+9/14 PzαB | 2µL | 20µL | 22µL | CP |
9/13 pSB2C3+9/14 PkaiBC | 2µL | 20µL | 22µL | CP |
9/13 RBS-GFP-DT+9/14 PkaiBC | 2µL | 20µL | 22µL | CP |
Plac+RBS-lysis1-DT | 2µL | 20µL | 22µL | Amp |
Plac+RBS-lysis2-DT | 2µL | 20µL | 22µL | Amp |
Plac+RBS-lysis2-DT | 2µL | 20µL | 22µL | Amp |
PCR
genome DNA | temp | Big Dye | 5x buffer | primer | MilliQ | total |
---|---|---|---|---|---|---|
9/6 PT181 attenuator(VR) | 3.9 | 0.5 | 1.75 | 0.5 | 3.85 | 10.5 |
8/17 RBS lacZα-DT | 2.1 | 0.5 | 1.75 | 0.5 | 3.85 | 10.5 |
9/6 PT181 attenuator(VR) | 3.9 | 0.5 | 1.75 | 0.5 | 3.85 | 10.5 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
96°C | 50°C | 60°C | 4°C | -- |
2min | 13s | 5s | 4min | 30cycle |