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Template:Kyoto/Notebook/Sep 24
From 2013.igem.org
(Difference between revisions)
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<span class="author">Hirano</span> | <span class="author">Hirano</span> | ||
{| class="wikitable" | {| class="wikitable" | ||
| - | !state||colspan="2"|Vector||colspan="2"|Inserter1||colspan="2"|Inserter2||colspan="2"|Inserter3||colspan="2"|Inserter4 | + | !state||colspan="2"|Vector||colspan="2"|Inserter1||colspan="2"|Inserter2||colspan="2"|Inserter3||colspan="2"|Inserter4||(NEB)T4ligase||(NEB)Buffer||milliQ||total |
|- | |- | ||
| - | |||RBS-GFP-DT(pSB1C3)||1µL||tetR-aptamer12-1R-DT(1-3)||1 µL||Ptet(2-S)||0.3µL||Pcon-tetR-aptamer12-1R-DT(3-2A)||1.1µL||Plac(E-1A)||0.5µL | + | |||RBS-GFP-DT(pSB1C3)||1µL||tetR-aptamer12-1R-DT(1-3)||1 µL||Ptet(2-S)||0.3µL||Pcon-tetR-aptamer12-1R-DT(3-2A)||1.1µL||Plac(E-1A)||0.5µL||0.5µL||0.5µL||0.2 µL||5µL |
|- | |- | ||
| - | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-pT181attenuator(E-1A)||1 µL||Pcon-tetR-aptamer12-1R-DT(1-2A)||1µL||Ptet(2-S)||0.3µL | + | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-pT181attenuator(E-1A)||1 µL||Pcon-tetR-aptamer12-1R-DT(1-2A)||1µL||Ptet(2-S)||0.3µL||-- ||-- ||0.5µL||0.5µL||0.7 µL||5µL |
|- | |- | ||
| - | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-tetRaptamer12-1R-DT(E-1A)||2µL||Pcon-tetRaptamer12-1R-DT(1-2A)||1µL||Ptet(2-S)||0.3µL | + | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-tetRaptamer12-1R-DT(E-1A)||2µL||Pcon-tetRaptamer12-1R-DT(1-2A)||1µL||Ptet(2-S)||0.3µL||--||--||0.5µL||0.5µL||0 µL||5.3µL |
|- | |- | ||
| - | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-spinach-DT(E-1A)||3µL||Pcon-tetRaptamer12-1R-DT(1-2A)||1µL||Ptet(2-S)||0.3µL||--||-- | + | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-spinach-DT(E-1A)||3µL||Pcon-tetRaptamer12-1R-DT(1-2A)||1µL||Ptet(2-S)||0.3µL||--||--|0.5µL||0.5µL||0 µL||6.3µL |
|- | |- | ||
| - | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-pT181antisense-DT(E-1A)||0.5µL||DT(1-3)||2µL||Ptet(2-S)||0.3µL||Pcon-tetRaptamer12-1R-DT(3-2A)||1.1µL | + | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-pT181antisense-DT(E-1A)||0.5µL||DT(1-3)||2µL||Ptet(2-S)||0.3µL||Pcon-tetRaptamer12-1R-DT(3-2A)||1.1µL||0.5µL||0.5µL||0.1 µL||5µL |
|- | |- | ||
| - | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-pT181attenuator(E-1A)||1µL||tetRaptamer12-1R-DT(1-3)||1µL||Ptet(2-S)||0.3µL||Pcon-tetRaptamer12-1R-DT(3-2A)||1.1µL | + | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-pT181attenuator(E-1A)||1µL||tetRaptamer12-1R-DT(1-3)||1µL||Ptet(2-S)||0.3µL||Pcon-tetRaptamer12-1R-DT(3-2A)||1.1µL||0.5µL||0.5µL||0µL||5.4µL |
|- | |- | ||
| - | |||RBS-GFP-DT(pSB1C3)||1µL||Ptet(2-S)||0.3µL||Pcon-tetRaptamer12-1R-DT(E-2A)||1µL | + | |||RBS-GFP-DT(pSB1C3)||1µL||Ptet(2-S)||0.3µL||Pcon-tetRaptamer12-1R-DT(E-2A)||1µL||--||--||--||--||0.5µL||0.5µL||1.7µL||5µL |
|- | |- | ||
| - | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-GFP-DT(2-SA)||0.5µL||Pcon-tetRaptamer12-1R-DT(E-2A)|1µL | + | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-GFP-DT(2-SA)||0.5µL||Pcon-tetRaptamer12-1R-DT(E-2A)|1µL||--||--||--||--||--||0.5µL||0.5µL||1.5µL||5µL |
|- | |- | ||
| - | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-tetRaptamer12-1R-DT(E-1A)||2µL||Pcon-pT181attenuator(1-SA)||2.1µL | + | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-tetRaptamer12-1R-DT(E-1A)||2µL||Pcon-pT181attenuator(1-SA)||2.1µL||--||--||--||--||0.5µL||0.5µL||0µL||6.1µL |
|- | |- | ||
| - | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-pT181attenuator-DT(E-1A)||1µL||Pcon-pT181attenuator(1-SA)||2.1µL | + | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-pT181attenuator-DT(E-1A)||1µL||Pcon-pT181attenuator(1-SA)||2.1µL||--||--||--||--||0.5µL||0.5µL||0µL||5.1µL |
|- | |- | ||
| - | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-tetR12-1R-DT(E-1A)||2µL||Pcon-GFP-DT(2-SA)||0.5µL||spinach-DT(3-2)||1µL||Ptet-pT181antisense(1-3)||1µL | + | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-tetR12-1R-DT(E-1A)||2µL||Pcon-GFP-DT(2-SA)||0.5µL||spinach-DT(3-2)||1µL||Ptet-pT181antisense(1-3)||1µL||0.5µL||0.5µL||0µL||6.5µL |
|- | |- | ||
| - | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-tetRaptamer12-1R-DT(E-1A)||2µL||Pcon-pT181antisense(1-2A)||0.7µL||Pcon-pT181attenuator(2-SA)||2.1µL | + | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-tetRaptamer12-1R-DT(E-1A)||2µL||Pcon-pT181antisense(1-2A)||0.7µL||Pcon-pT181attenuator(2-SA)||2.1µL||--||--||0.5µL||0.5µL||0µL||6.8µL |
|- | |- | ||
| - | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-tetRaptamer12-1R-DT(E-1A)||2µL||Pcon-pT181attenuator(2-SA)||2.1µL||Spinach-DT(3-2)||1µL||Ptet-pT181antisense(1-3)||1µL | + | |||RBS-GFP-DT(pSB1C3)||1µL||Pcon-tetRaptamer12-1R-DT(E-1A)||2µL||Pcon-pT181attenuator(2-SA)||2.1µL||Spinach-DT(3-2)||1µL||Ptet-pT181antisense(1-3)||1µL||0.5µL||0.5µL||0µL||8.1µL |
|} | |} | ||
<br>Concentrate total (more than 5µL) by evaporater until volume become less than 5 µL<br> | <br>Concentrate total (more than 5µL) by evaporater until volume become less than 5 µL<br> | ||
</div> | </div> | ||
Revision as of 16:11, 26 September 2013
Sep 24
Electrophoresis
| Lane | Sample |
|---|---|
| 1 | 9/20 Pcon-pT181aptamer12-1R-DT 1 |
| 2 | 9/20 Pcon-pT181aptamer12-1R-DT 2 |
| 3 | 9/20 Pcon-pT181aptamer12-1R-DT 3 |
| 4 | 9/20 Pcon-pT181aptamer12-1R-DT 4 |
| 5 | 9/20 Pcon-pT181aptamer12-1R-DT 5 |
| 6 | 9/20 Pcon-pT181aptamer12-1R-DT 6 |
| 7 | 9/20 Pcon-pT181aptamer12-1R-DT 7 |
| 8 | 9/20 Pcon-pT181aptamer12-1R-DT 8 |
| 9 | 100bp ladder |
| 10 | 9/22 Pcon-pT181attenuator-aptamer12-1R-DT 1 |
| 11 | 9/22 Pcon-pT181attenuator-aptamer12-1R-DT 2 |
| 12 | 9/22 Pcon-pT181attenuator-aptamer12-1R-DT 3 |
| 13 | 9/22 Pcon-pT181attenuator-aptamer12-1R-DT 4 |
| 14 | 9/20 Plac-aptamer12-1R-DT 1 |
| 15 | 9/20 Plac-aptamer12-1R-DT 2 |
| 16 | 9/20 Plac-aptamer12-1R-DT 3 |
| 17 | 9/20 Plac-aptamer12-1R-DT 4 |
Transformation
| Name | Sample | Competent Cells | Total | Plate |
|---|---|---|---|---|
| 9/23 pSB1C3-pT181attenuator | 2 µL | 20 µL | 22 µL | LB (+CP) |
| RBS-GFP-DT+Pcon-pT181attenuator | 2 µL | 20 µL | 22 µL | LB (+CP) |
| 9/23 Plac-+spinach-DT | 2 µL | 20 µL | 22 µL | LB (+Amp) |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT | 2 µL | 20 µL | 22 µL | LB (+Amp) |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT | 2 µL | 20 µL | 22 µL | LB (+Amp) |
| 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT | 2 µL | 20 µL | 22 µL | LB (+Amp) |
| 9/23 Pcon-pT181antisense-DT | 2 µL | 20 µL | 22 µL | LB (+CP) |
| 9/23 Pcon-pT181attenuator-RBS-GFP-DT | 2 µL | 20 µL | 22 µL | LB (+CP) |
| 9/23 Pcon-pT181attenuator-aptamer12-1R-DT | 2 µL | 20 µL | 22 µL | LB (+CP) |
| PUC19 | 2 µL | 20 µL | 22 µL | LB (+Amp) |
| Ptet-RBS-lacZα-DT | 2 µL | 20 µL | 22 µL | LB (+CP) |
| pSB1C3 | 2 µL | 20 µL | 22 µL | LB (+CP) |
Liquid Culture
| Sample | medium |
|---|---|
| aptamer12-1R-DT(Master plate) | LB(+CP) |
Transformation
| Name | Sample | Competent Cells | Total | Plate |
|---|---|---|---|---|
| 9/23 Pcon-attenuator-aptamer12-1R-DT | 2 µL | 20 µL | 22 µL | LB (+CP) |
| 9/23 pT181attenuator(1C3) | 2 µL | 20 µL | 22 µL | LB (+CP) |
| 9/23 Pcon-pT181antisense-DT | 2 µL | 20 µL | 22 µL | LB (+CP) |
| 9/23 Pcon-pT181attenuator-RBS--GFP-DT | 2 µL | 20 µL | 22 µL | LB (+CP) |
Liquid Culture
| Sample | medium |
|---|---|
| pSB1C3 (Master plate-1) | LB(+CP) |
Restriction Enzyme Digestion
| 8/21 pT181attenuator (330 µg/mL) | XbaI | PstI | buffer | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 2 cuts | 6.1 | 1 | 1 | 3 | 3 | 15.9 | 30 |
| NC | 0.3 | 0 | 0 | 1 | 1 | 7.7 | 10 |
| 9/22 Pcon-pT181attenuator (368.3 µg/mL) | EcoRI | SpeI | buffer | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 2 cuts | 5.4 | 1 | 1 | 3 | 0 | 19.6 | 30 |
| NC | 0.3 | 0 | 0 | 1 | 0 | 8.7 | 10 |
| 9/16 pSB1C3 | EcoRI | SpeI | buffer | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 2 cuts | 18 | 1 | 1 | 3 | 0 | 7.0 | 30 |
| NC | 0.9 | 0 | 0 | 1 | 0 | 8.1 | 10 |
Electrophoresis
| Lane | Sample | Enzyme1 | Enzyme2 |
|---|---|---|---|
| 1 | 1kb ladder | - | - |
| 2 | pT181attenuator | XbaI | PstI |
| 3 | pT181attenuator | -- | -- |
| 4 | Pcon-pT181attenuator | EcoRI | SpeI |
| 5 | Pcon-pT181attenuator | -- | -- |
| 6 | pSB1C3 | EcoRI | SpeI |
| 7 | pSB1C3 | -- | -- |
Miniprep
| DNA | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| 9/24 aptamer12-1R-DT | 144.7 | 1.03 | 1.21 |
Gel Extraction
| Lane | DNA | Enzyme1 | Enzyme2 |
|---|---|---|---|
| 1 | 100kb ladder | -- | -- |
| 2 | pT181attenuator | XbaI | PstI |
| 3 | pT181attenuator | XbaI | PstI |
| 4 | pT181attenuator | XbaI | PstI |
| 5 | -- | -- | -- |
| 6 | Pcon-pT181attenuator | EcoRI | SpeI |
| 7 | Pcon-pT181attenuator | EcoRI | SpeI |
| 8 | Pcon-pT181attenuator | EcoRI | SpeI |
| 9 | -- | -- | -- |
| 10 | pSB1C3 | EcoRI | SpeI |
| 11 | pSB1C3 | EcoRI | SpeI |
| 12 | pSB1C3 | EcoRI | SpeI |
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| pT181attenuator(XbaI&PstI) | 10.1 | 2.02 | 0.43 |
| Pcon-pT181attenuator(EcoRI&SpeI) | 10.3 | 1.81 | 0.32 |
| pSB1C3(EcoRI&SpeI) | 10.0 | 2.81 | 0.02 |
Colony PCR
| Sample | base pair |
|---|---|
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 1 | 1414 |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2 | 1414 |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 3 | 1414 |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 4 | 1414 |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 5 | 1414 |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 6 | 1414 |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 7 | 1414 |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 8 | 1414 |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 1 | 1631 |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2 | 1631 |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 3 | 1631 |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 4 | 1631 |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 5 | 1631 |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 6 | 1631 |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 7 | 1631 |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8 | 1631 |
| 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 1 | 2042 |
| 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2 | 2042 |
| 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 3 | 2042 |
| 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 4 | 2042 |
| 9/23 Pcon-pT181antisense-DT 1 | 508 |
| 9/23 Pcon-pT181antisense-DT 2 | 508 |
| 9/23 Pcon-pT181antisense-DT 3 | 508 |
| 9/23 Pcon-pT181antisense-DT 4 | 508 |
| 9/23 Pcon-pT181antisense-DT 5 | 508 |
| 9/23 Pcon-pT181antisense-DT 6 | 508 |
| 9/23 Pcon-pT181antisense-DT 7 | 508 |
| 9/23 Pcon-pT181antisense-DT 8 | 508 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94 °C | 94 °C | 55 °C | 68 °C | -- |
| 5 min | 30 sec | 30 sec | 2min | 30 cycles |
| Lane | Sample |
|---|---|
| 1 | 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 1 |
| 2 | 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2 |
| 3 | 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 3 |
| 4 | 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 4 |
| 5 | 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 5 |
| 6 | 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 6 |
| 7 | 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 7 |
| 8 | 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 8 |
| 9 | 1kb ladder |
| 10 | 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 1 |
| 11 | 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2 |
| 12 | 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 3 |
| 13 | 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 4 |
| 14 | 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 5 |
| 15 | 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 6 |
| 16 | 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 7 |
| 17 | 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8 |
</div>
| Lane | Sample |
|---|---|
| 1 | 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 1 |
| 2 | 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2 |
| 3 | 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 3 |
| 4 | 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 4 |
| 5 | 9/23 Pcon-pT181antisense-DT 1 |
| 6 | 9/23 Pcon-pT181antisense-DT 2 |
| 7 | 1kb ladder |
| 8 | 9/23 Pcon-pT181antisense-DT 3 |
| 9 | 9/23 Pcon-pT181antisense-DT 4 |
| 10 | 9/23 Pcon-pT181antisense-DT 5 |
| 11 | 9/23 Pcon-pT181antisense-DT 6 |
| 12 | 9/23 Pcon-pT181antisense-DT 7 |
| 13 | 9/23 Pcon-pT181antisense-DT 8 |
| 14 | -- |
</div>
Restriction Enzyme Digestion
| 9/24 aptamer12-1R-DT | EcoRI | XbaI | buffer | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 2cuts | 17.5µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 4.5µL | 30µL |
| NC | 0.9µL | 0µL | 0µL | 1.0µL | 1.0µL | 7.1µL | 10µL |
| 9/20 Ptet | SpeI | PstI | buffer | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 2cuts | 9.5µL | 1.0µL | 1.0µL | 3.0µL | 0µL | 15.5µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 1.0µL | 0µL | 8.5µL | 10µL |
| 9/22 RBS-GFP-DT | EcoRI | SpeI | buffer | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 2cuts | 3.8µL | 1.0µL | 1.0µL | 3.0µL | 0µL | 21.2µL | 30µL |
| NC | 0.2µL | 0µL | 0µL | 1.0µL | 0µL | 8.8µL | 10µL |
| 9/22 Pcon-RBS-GFP-DT | EcoRI | XbaI | buffer | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 2cuts | 2.4µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 19.6µL | 30µL |
| NC | 0.1µL | 0µL | 0µL | 1.0µL | 1.0µL | 7.9µL | 10µL |
| 9/22 Pcon-pT181attenuator 1 | EcoRI | XbaI | buffer | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 2cuts | 5.4µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 16.6µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 1.0µL | 1.0µL | 7.7µL | 10µL |
| 9/22 RBS-GFP-DT | XbaI | PstI | buffer | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 2cuts | 3.8µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 18.2µL | 30µL |
| NC | 0.2µL | 0µL | 0µL | 1.0µL | 1.0µL | 7.8µL | 10µL |
Ligation
| state | Vector | Inserter | Ligation High ver.2 | ||
|---|---|---|---|---|---|
| 9/22 aptamer12-1R-DT(EcoRI&XbaI) | 1.4 µL | 9/24 Pcon-pT181attenuator(EcoRI&SpeI) | 26 µL | 3.5 µL | |
| 9/4 Ptet(SpeI&PstI) | 2.0 µL | 9/21 RBS-GFP-DT(XbaI&PstI) | 9.0 µL | 3.5 µL | |
- incubated at 37 °C for 1 hour.
colony PCR
| Sample | base pair |
|---|---|
| 9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 5 | 2042bp |
| 9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 6 | 2042bp |
| 9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 7 | 2042bp |
| 9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 8 | 2042bp |
| 9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 9 | 2042bp |
| 9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 10 | 2042bp |
| 9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 11 | 2042bp |
| 9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 12 | 2042bp |
| 9/23 Pcon-pT181antisense-DT 9 | 508bp |
| 9/23 Pcon-pT181antisense-DT 10 | 508bp |
| 9/23 Pcon-pT181antisense-DT 11 | 508bp |
| 9/23 Pcon-pT181antisense-DT 12 | 508bp |
| 9/23 Pcon-pT181antisense-DT 13 | 508bp |
| 9/24 Pcon-pT181attenuator-RBS-GFP-DT 1 | 1527bp |
| 9/24 Pcon-pT181attenuator-RBS-GFP-DT 2 | 1527bp |
| 9/24 Pcon-pT181attenuator-RBS-GFP-DT 3 | 1527bp |
| 9/24 Pcon-pT181attenuator-RBS-GFP-DT 4 | 1527bp |
| 9/24 Pcon-pT181attenuator-aptamer12-1R-DT 1 | 859bp |
| 9/24 Pcon-pT181attenuator-aptamer12-1R-DT 2 | 859bp |
| 9/24 Pcon-pT181attenuator-aptamer12-1R-DT 3 | 859bp |
| 9/24 Pcon-pT181attenuator-aptamer12-1R-DT 4 | 859bp |
| 9/24 pT181attenuator(pSB1C3) 1 | 390bp |
| 9/24 pT181attenuator(pSB1C3) 2 | 390bp |
| 9/24 pT181attenuator(pSB1C3) 3 | 390bp |
| 9/24 pT181attenuator(pSB1C3) 4 | 390bp |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 2min | 30cycle |
Miniprep
| DNA | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| pSB1C3 | 183.9 | 1.91 | 1.59 |
| Pcon-pT181attenuator-aptamer12-1R-DT | 231.7 | 1.82 | 2.35 |
| Plac-aptamer12-1R-DT | 208.9 | 1.98 | 1.79 |
Electrophoresis
| Lane | Sample | Enzyme1 | Enzyme2 |
|---|---|---|---|
| 1 | 1kb ladder | -- | -- |
| 2 | 9/24 aptamer12-1R-DT | EcoRI | XbaI |
| 3 | 9/24 aptamer12-1R-DT | -- | -- |
| 4 | 9/24 Ptet | SpeI | PstI |
| 5 | 9/24 Ptet | -- | -- |
| 6 | 9/24 RBS-GFP-DT | EcoRI | SpeI |
| 7 | 9/24 RBS-GFP-DT | -- | -- |
| 8 | 9/24 Pcon-RBS-GFP-DT | EcoRI | XbaI |
| 8 | 9/24 Pcon-RBS-GFP-DT | -- | -- |
| 9 | 9/24 Pcon-RBS-GFP-DT | -- | -- |
| 10 | 9/24 Pcon-pT181attenuator 1 | EcoRI | XbaI |
| 11 | 9/24 Pcon-pT181attenuator 1 | -- | -- |
| 12 | 9/24 RBS-GFP-DT | XbaI | PstI |
| 13 | 9/24 RBS-GFP-DT | -- | -- |
| 14 | 1kb ladder | -- | -- |
Gel Extraction
1
| Lane | DNA | Enzyme1 | Enzyme2 |
|---|---|---|---|
| 1 | 1kb ladder | -- | -- |
| 2 | aptamer12-1R-DT | EcoRI | XbaI |
| 3 | aptamer12-1R-DT | EcoRI | XbaI |
| 4 | aptamer12-1R-DT | EcoRI | XbaI |
| 5 | -- | -- | -- |
| 6 | Ptet | SpeI | PstI |
| 7 | Ptet | SpeI | PstI |
| 8 | Ptet | SpeI | PstI |
| 9 | -- | -- | -- |
| 10 | RBS-GFP-DT | EcoRI | SpeI |
| 11 | RBS-GFP-DT | EcoRI | SpeI |
| 12 | RBS-GFP-DT | EcoRI | SpeI |
| Lane | DNA | Enzyme1 | Enzyme2 |
|---|---|---|---|
| 1 | 1kb ladder | -- | -- |
| 2 | Pcon-RBS-GFP-DT | EcoRI | XbaI |
| 3 | Pcon-RBS-GFP-DT | EcoRI | XbaI |
| 4 | Pcon-RBS-GFP-DT | EcoRI | XbaI |
| 5 | -- | -- | -- |
| 6 | Pcon-pT181attenuator | EcoRI | XbaI |
| 7 | Pcon-pT181attenuator | EcoRI | XbaI |
| 8 | Pcon-pT181attenuator | EcoRI | XbaI |
| 9 | -- | -- | -- |
| 10 | RBS-GFP-DT | XbaI | PstI |
| 11 | RBS-GFP-DT | XbaI | PstI |
| 12 | RBS-GFP-DT | XbaI | PstI |
Electrophoresis
1
| Lane | Sample |
|---|---|
| 1 | 1kb ladder |
| 2 | Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 5 |
| 3 | Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 6 |
| 4 | Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 7 |
| 5 | Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 8 |
| 6 | Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 9 |
| 7 | Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 10 |
| 8 | 1kb ladder |
2
| Lane | Sample |
|---|---|
| 1 | 1kb ladder |
| 2 | Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 11 |
| 3 | Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 12 |
| 4 | Pcon-pT181attenuator-RBS-GFP-DT 1 |
| 5 | Pcon-pT181attenuator-RBS-GFP-DT 2 |
| 6 | Pcon-pT181attenuator-RBS-GFP-DT 3 |
| 7 | Pcon-pT181attenuator-RBS-GFP-DT 4 |
| 8 | 1kb ladder |
3
| Lane | Sample |
|---|---|
| 1 | Pcon-pT181antisense-DT 9 |
| 2 | Pcon-pT181antisense-DT 10 |
| 3 | Pcon-pT181antisense-DT 11 |
| 4 | Pcon-pT181antisense-DT 12 |
| 5 | Pcon-pT181antisense-DT 13 |
| 6 | 100bp ladder |
| 7 | Pcon-pT181attenuator-aptamer12-1R-DT 1 |
| 8 | Pcon-pT181attenuator-aptamer12-1R-DT 2 |
4
| Lane | Sample |
|---|---|
| 1 | Pcon-pT181attenuator-aptamer12-1R-DT 3 |
| 2 | Pcon-pT181attenuator-aptamer12-1R-DT 4 |
| 3 | 100bp ladder |
| 4 | pT181attenuator(pSB1C3) 1 |
| 5 | pT181attenuator(pSB1C3) 2 |
| 6 | pT181attenuator(pSB1C3) 3 |
| 7 | pT181attenuator(pSB1C3) 4 |
PCR
| genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | F primer | R primer | KOD-plus | MilliQ | total |
|---|---|---|---|---|---|---|---|---|---|
| Pcon antisense(1C3-GGE-fwd,1A2-GG2-rev) | 1µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 0.5µL | 15.5 | 25µL |
| DT-(1C3-GG1-fwd,1C3-GG2-rev) | 1µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 0.5µL | 15.5 | 25µL |
| Spinach-DT-(1C3-GG1-fwd,1C3-GG2-rev) | 1µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 0.5µL | 15.5 | 25µL |
| Pcon-antisense-spinach-DT(1C3-GGE-fwd,1A2-GG1-rev) | 1µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 0.5µL | 15.5 | 25µL |
| Ptet(1C3-GGE-fwd,1C3-GG0-rev) | 1µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 0.5µL | 15.5 | 25µL |
| Pcon attenuator(1C3-GGE-fwd,1C3-GG0-rev) | 1µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 0.5µL | 15.5 | 25µL |
| DT(1C3-GG2-fwd,1C3-GG1-rev) | 1µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 0.5µL | 15.5 | 25µL |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10sec | 30sec | 36sec | 30 |
Ligation
| state | Vector | Inserter1 | Inserter2 | Inserter3 | Inserter4 | (NEB)T4ligase | (NEB)Buffer | milliQ | total | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| RBS-GFP-DT(pSB1C3) | 1µL | tetR-aptamer12-1R-DT(1-3) | 1 µL | Ptet(2-S) | 0.3µL | Pcon-tetR-aptamer12-1R-DT(3-2A) | 1.1µL | Plac(E-1A) | 0.5µL | 0.5µL | 0.5µL | 0.2 µL | 5µL | |
| RBS-GFP-DT(pSB1C3) | 1µL | Pcon-pT181attenuator(E-1A) | 1 µL | Pcon-tetR-aptamer12-1R-DT(1-2A) | 1µL | Ptet(2-S) | 0.3µL | -- | -- | 0.5µL | 0.5µL | 0.7 µL | 5µL | |
| RBS-GFP-DT(pSB1C3) | 1µL | Pcon-tetRaptamer12-1R-DT(E-1A) | 2µL | Pcon-tetRaptamer12-1R-DT(1-2A) | 1µL | Ptet(2-S) | 0.3µL | -- | -- | 0.5µL | 0.5µL | 0 µL | 5.3µL | |
| RBS-GFP-DT(pSB1C3) | 1µL | Pcon-spinach-DT(E-1A) | 3µL | Pcon-tetRaptamer12-1R-DT(1-2A) | 1µL | Ptet(2-S) | 0.3µL | -- | 0.5µL | 0.5µL | 0 µL | 6.3µL | ||
| RBS-GFP-DT(pSB1C3) | 1µL | Pcon-pT181antisense-DT(E-1A) | 0.5µL | DT(1-3) | 2µL | Ptet(2-S) | 0.3µL | Pcon-tetRaptamer12-1R-DT(3-2A) | 1.1µL | 0.5µL | 0.5µL | 0.1 µL | 5µL | |
| RBS-GFP-DT(pSB1C3) | 1µL | Pcon-pT181attenuator(E-1A) | 1µL | tetRaptamer12-1R-DT(1-3) | 1µL | Ptet(2-S) | 0.3µL | Pcon-tetRaptamer12-1R-DT(3-2A) | 1.1µL | 0.5µL | 0.5µL | 0µL | 5.4µL | |
| RBS-GFP-DT(pSB1C3) | 1µL | Ptet(2-S) | 0.3µL | Pcon-tetRaptamer12-1R-DT(E-2A) | 1µL | -- | -- | -- | -- | 0.5µL | 0.5µL | 1.7µL | 5µL | |
| RBS-GFP-DT(pSB1C3) | 1µL | Pcon-GFP-DT(2-SA) | 0.5µL | 1µL | -- | -- | -- | -- | -- | 0.5µL | 0.5µL | 1.5µL | 5µL | |
| RBS-GFP-DT(pSB1C3) | 1µL | Pcon-tetRaptamer12-1R-DT(E-1A) | 2µL | Pcon-pT181attenuator(1-SA) | 2.1µL | -- | -- | -- | -- | 0.5µL | 0.5µL | 0µL | 6.1µL | |
| RBS-GFP-DT(pSB1C3) | 1µL | Pcon-pT181attenuator-DT(E-1A) | 1µL | Pcon-pT181attenuator(1-SA) | 2.1µL | -- | -- | -- | -- | 0.5µL | 0.5µL | 0µL | 5.1µL | |
| RBS-GFP-DT(pSB1C3) | 1µL | Pcon-tetR12-1R-DT(E-1A) | 2µL | Pcon-GFP-DT(2-SA) | 0.5µL | spinach-DT(3-2) | 1µL | Ptet-pT181antisense(1-3) | 1µL | 0.5µL | 0.5µL | 0µL | 6.5µL | |
| RBS-GFP-DT(pSB1C3) | 1µL | Pcon-tetRaptamer12-1R-DT(E-1A) | 2µL | Pcon-pT181antisense(1-2A) | 0.7µL | Pcon-pT181attenuator(2-SA) | 2.1µL | -- | -- | 0.5µL | 0.5µL | 0µL | 6.8µL | |
| RBS-GFP-DT(pSB1C3) | 1µL | Pcon-tetRaptamer12-1R-DT(E-1A) | 2µL | Pcon-pT181attenuator(2-SA) | 2.1µL | Spinach-DT(3-2) | 1µL | Ptet-pT181antisense(1-3) | 1µL | 0.5µL | 0.5µL | 0µL | 8.1µL | |
Concentrate total (more than 5µL) by evaporater until volume become less than 5 µL
