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Template:Kyoto/Notebook/Sep 17
From 2013.igem.org
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|Spinach-DT||LB medium(+CP) | |Spinach-DT||LB medium(+CP) | ||
|- | |- | ||
| - | |Pcon- | + | |Pcon-Spinach-DT||LB medium(+Amp) |
| + | |- | ||
| + | |Pcon-pT181 antisense-Spinach-DT||LB medium(+Amp) | ||
|} | |} | ||
</div> | </div> | ||
Revision as of 11:46, 24 September 2013
Contents |
Sep 17
Liquid Culture
| Sample | medium |
|---|---|
| Spinach-DT | LB medium(+CP) |
| Pcon-Spinach-DT | LB medium(+Amp) |
| Pcon-pT181 antisense-Spinach-DT | LB medium(+Amp) |
PCR
| Pcon-tet ap-DT(VF) | BigDye | 5x buffer | primer(VF) | MilliQ | total |
|---|---|---|---|---|---|
| 1.3 | 0.5 | 1.75 | 0.5 | 0.45 | 10.5 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 96°C | 96°C | 50°C | 60°C | -- |
| 2m | 10s | 5s | 4m | 30cycles |
| Pcon-tet ap-DT(VR) | BigDye | 5x buffer | primer(VR) | MilliQ | total |
|---|---|---|---|---|---|
| 1.3 | 0.5 | 1.75 | 0.5 | 0.45 | 10.5 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 96°C | 96°C | 50°C | 60°C | -- |
| 2m | 10s | 5s | 4m | 30cycles |
Electrophoresis
| Lane | Sample | Enzyme1 | Enzyme2 |
|---|---|---|---|
| 1 | 1kbpladder | -- | -- |
| 2 | Pcon-RBS-tetR-DT | XbaI | PstI |
| 3 | Pcon-RBS-tetR-DT | -- | -- |
| 4 | pSB4K5X7 | EcoRI | XbaI |
| 5 | pSB4K5X7 | -- | -- |
| 6 | PSB1C3 | EcoRI | SpeI |
| 7 | PSB1C3 | EcoRI | PstI |
| 8 | PSB1C3 | -- | -- |
| 9 | pT181attenuator | -- | -- |
| 10 | pT181attenuator | -- | -- |
| 11 | 1kbpladder | -- | -- |
Ligation
| state | Vector | Inserter | Ligation High ver.2 | ||
|---|---|---|---|---|---|
| experiment | 8/28 Plux-(SpeI&PstI) | 5.0µg | 9/13 RBS-lysis1-DT-(XbaI&PstI) | 3.5µg | 3.5µg |
| experiment | 8/28 Plux-(SpeI&PstI) | 5.0micro;g | 9/13 RBS-lysis2-DT-(XbaI&PstI) | 3.6µg | 3.5µg |
| experiment | 8/28 Plux-(SpeI&PstI) | 11.0µg | 9/13 RBS-lysis3-DT-(XbaI&PstI) | 9.5µg | 3.5µg |
| experiment | 9/14 Plac-(SpeI&PstI) | 2.4µg | 9/16 PT181-attenuator-DT-(XbaI&PstI) | 4.8µg | 3.5µg |
| experiment | 9/14 Plac-(SpeI&PstI) | 2.4µg | 9/3 PT181-antisense-(XbaI&PstI) | 2.0µg | 2.2µg |
| experiment | 9/14 Plac-(SpeI&PstI) | 2.4µg | 9/10 aptamer12-1R-DT-(XbaI&PstI) | 1.8µg | 2.1µg |
| experiment | 9/13 pSB1C3-(XbaI&PstI) | 2.0µg | 9/15 Pcon-PT181 antisense-spinach-DT-(XbaI&PstI) | 12.0µg | 3.5µg |
| experiment | 9/16 pSB1C3-(EcoRI&SpeI) | 1.0µg | 9/15 Pcon-spinach-DT-(EcoRI&SpeI) | 4.1µg | 2.0µg |
| experiment | 9/16 pSB1C3-(EcoRI&SpeI) | 1.0µg | 9/15 Pcon-RBS-tetR-DT-(EcoRI&SpeI) | 2.0µg | 1.5µg |
| experiment | 9/16 pSB1C3-(EcoRI&SpeI) | 1.0µg | 9/15 Pcon-PT181-attenuator-DT-(EcoRI&SpeI) | 2.4µg | 1.7µg |
| experiment | 9/16 pSB1C3-(EcoRI&SpeI) | 1.0µg | 9/15 Pcon-aptamer12-112-DT-(EcoRI&SpeI) | 17.0µg | 1.5µg |
| experiment | 9/16 pSB1C3-(XbaI&PstI) | 1.0µg | 9/15 Pcon-aptamer12-112-DT-(EcoRI&SpeI) | 4.4µg | 3.5µg |
Gel Extraction
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 1kbpladder | -- |
| 2 | Pt181 attenuator -(2) | EcoRI+SpeI |
| 3 | ||
| 4 |
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| PT181-attenuator EcoI+SpeI | 9.2 | 1.15 | 0.12 |
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 1kbpladder | -- |
| 2 | pSB1C3 | EcoI+SpeI |
| 3 | ||
| 4 |
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| pSB1C3 | 10.0 | 1.84 | 0.40 |
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 1kbpladder | -- |
| 2 | pSB4K5 | XbaI+PstI |
| 3 | ||
| 4 |
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| pSB4K5 | 7.6 | 1.81 | 0.32 |
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 1kbpladder | -- |
| 2 | Pcon-RBS-tetR-DT | XbaI+PstI |
| 3 | ||
| 4 |
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| Pcon-RBS-tetR-DT | 8.0 | 2.40 | 1.14 |
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 1kbpladder | -- |
| 2 | pSB1C3 | EcoRI+PstI |
| 3 | ||
| 4 |
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| pSB1C3 | 9.1 | 2.17 | 0.35 |
Mutation PCR
| pC KaiABC(100pg) | Prime STAR(2x) | Primer(KaiA-Bsai MuT-Fwd) | Primer(KaiA-Bsai MuT-Rev) | MilliQ | total |
|---|---|---|---|---|---|
| 1.0 | 25.0 | 1.0 | 1.0 | 22.0 | 50.0 |
| 1.5 | 25.0 | 1.0 | 1.0 | 21.5 | 50.0 |
| 2.0 | 25.0 | 1.0 | 1.0 | 21.0 | 50.0 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 96°C | 98°C | 65°C | 72°C | -- |
| 5m | 10s | 15s | 40s | 30cycles |
