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Template:Kyoto/Notebook/Sep 21
From 2013.igem.org
Contents |
Sep 21
Electrophoresis
| Lane | Sample | Enzyme1 | Enzyme2 |
|---|---|---|---|
| 1 | Pcon-RBS-lacZα-DT | -- | -- |
| 2 | 1kbp ladder | -- | -- |
| 3 | pSB1C3+pT181 attenuator | -- | -- |
Liquid Culture
| Sample | medium |
|---|---|
| 9/2 spinach-DT-2 | -- |
| 8/16 Plux-4 | -- |
| 8/27 Plux-RBS-GFP-DT-1 | -- |
| 9/5 Pcon-pT181 attenuator-1 | -- |
Master Plate
| Number | Use LB plate |
|---|---|
| 1 | 8/27 Pλ-luxI-1(+Amp) |
| 2 | 9/10 Plac(BBa-R0011)-3(+Amp) |
| 3 | 9/5 Pcon-RBS-tetR-DT-1(+Amp) |
| 4 | 9/5 Pcon-pT181 antisense-1(+Amp) |
| 5 | 9/15 Pcon-pT181 attenuator(+Amp) |
| 6 | 9/- Pcon-apt12_1R-DT-1(+Amp) |
| 7 | 9/5 Pcon-spinach-DT-3(+Amp) |
| 8 | 8/27 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT(+Amp) |
| Number | Use LB plate |
|---|---|
| 1 | 9/4 pT181 antisense(pSB1C3)-1(+CP) |
| 2 | 9/11 Fusion1 antisense(pSB1C3)-4(+CP) |
| 3 | 9/11 Fusion6 antisense(pSB1C3)-1(+CP) |
| 4 | 9/4 pT181 attenuator(pSB1C3)-1(+CP) |
| 5 | 9/10 Fusion1 attenuator(pSB1C3)-1(+CP) |
| 6 | 9/11 Fusion3n2 attenuator(pSB1C3)-1(+CP) |
| 7 | 9/4 aptamer12_1R(pSB1C3)-1(+CP) |
| 8 | 9/11 aptamer12_P(pSB1C3)-3(+CP) |
| 9 | 9/11 aptamer12_1M(pSB1C3)-1(+CP) |
</div>
| Number | Use LB plate |
|---|---|
| 1 | 8/29 Pbad/araC-RBS-RFP-DT-1(+CP) |
| 2 | 9/4 RBS-lysis3-DT-3(+CP) |
| 3 | 9/10 Pcon-pT181 attenuator-DT-1(+CP) |
| 4 | 9/5 Ptet-pT181 antisense-1(+CP) |
| 5 | 9/4aptamer 12_1R-DT-1(+CP) |
| 6 | 9/5 Ptet-RBS-lacZα-DT-4(+CP) |
| 7 | 8/10 RBS-lacI-DT-2(+CP) |
| 8 | 8/29 Plux-RBS-GFP-DT-1(+CP) |
</div>
Restriction Enzyme Digestion
| 8/17 RBS-DT | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|---|---|
| 2cuts(XbaI&PstI) | 8.1 | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 13.9µL | 30µL |
| NC | 0.4 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |
| 8/17 RBS-DT | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|---|---|
| 2cut(EcoRI&XbaI) | 16.2 | 1µL | 1µL | 0µL | 0µL | 3µL | 3µL | 5.8µL | 30µL |
| 9/15 Pcon-pT181attenuator | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|---|---|
| 2cuts(EcoRI&PstI) | 12 | 1µL | 0µL | 0µL | 1µL | 3µL | 3µL | 10µL | 30µL |
| NC | 0.6 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.4µL | 10µL |
| 8/20 Pcon=RBS-GFP-DT-1 | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|---|---|
| 2cut(EcoRI&XbaI) | 6 | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 0µL | 14µL |
| NC | 0.3 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.3µL | 10µL |
Electrophoresis
| Lane | Sample | Enzyme1 | Enzyme2 |
|---|---|---|---|
| 1 | RBS-GFP-DT | XbaI | PstI |
| 2 | RBS-GFP-DT | -- | -- |
| 3 | RBS-GFP-DT | EcoRI | XbaI |
| 4 | 1kbp ladder | -- | -- |
| 5 | Pcon-pT181attenuator | SpeI | PstI |
| 6 | Pcon-pT181attenuator | -- | -- |
| 7 | Pcon-RBs-GFP-DT | EcoRI | XbaI |
| 8 | Pcon-RBs-GFP-DT | -- | -- |
PCR
| Sample | base pair |
|---|---|
| Pcon-apt12_1R-DT(E-1A) | 50 |
| Pcon-spinach-DT(E-1A) | 402 |
| Pcon-pT181 attenuator(E-1A) | 400 |
| DT(1-3) | 65 |
| tetR aptamer 12_1R-DT(1-3) | 283 |
| Pcon-pT181 attenuator(1-SA) | 303 |
| spinach-DT(2-1) | 338 |
| Pcon-pT181 attenuator(E-0) | 403 |
| Pcon-pT181 attenuator(2-SA) | 403 |
| spinach-DT(3-2) | 395 |
| Ptet-PT181 anisense | 400 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10s | 30s | 35s | 30cycles |
Gel Extraction
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 1kb ladder | -- |
| 3 | RBS-GFP-DT | XbaI&PstI |
| 4 | RBS-GFP-DT | XbaI&PstI |
| 5 | RBS-GFP-DT | XbaI&PstI |
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| RBS-GFP-DT(XbaI&PstI) | 14.8 | 1.74 | 0.47 |
| RBS-GFP-DT(EcoRI&SpeI) | 88.4 | 1.85 | 1.49 |
| Pcon-RBS-GFP-DT(EcoRI&XbaI) | 47.5 | 1.82 | 1.12 |
