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Template:Kyoto/Notebook/Sep 25
From 2013.igem.org
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===Liquid Culture=== | ===Liquid Culture=== | ||
<div class="experiment"> | <div class="experiment"> | ||
| - | <span class="author"> | + | <span class="author">Kojima</span> |
{| class="wikitable" | {| class="wikitable" | ||
!Sample||medium | !Sample||medium | ||
|- | |- | ||
| - | | | + | |DT ||- |
|- | |- | ||
| - | | | + | |pT181attenuator ||- |
| + | |- | ||
| + | |Pcon-RBS-tetR-DT ||- | ||
|} | |} | ||
</div> | </div> | ||
Revision as of 12:59, 26 September 2013
Sep 25
Colony PCR
| Sample | base pair |
|---|---|
| 9/23 Pcon-PT181 attenuator-aptamer12-1R-DT-(5~12) | 859 |
| 9/23 Pcon-PT181 attenuator-RBS-GFP-DT-(5~8) | 1527 |
| 9/23 Pcon-PT181 attenuator-pSB1C3-(5~8) | 601 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94 °C | 94 °C | 55 °C | 68 °C | -- |
| 2 min | 30 s | 30 s | 1min5s | 30 cycles |
Electrophoresis
| Lane | Sample | Enzyme1 | Enzyme2 |
|---|---|---|---|
| 1 | Pcon-PT181 attenuator-aptamer12-1R-DT-5 | -- | -- |
| 2 | Pcon-PT181 attenuator-aptamer12-1R-DT-6 | -- | -- |
| 3 | Pcon-PT181 attenuator-aptamer12-1R-DT-7 | -- | -- |
| 4 | Pcon-PT181 attenuator-aptamer12-1R-DT-8 | -- | -- |
| 5 | Pcon-PT181 attenuator-aptamer12-1R-DT-9 | -- | -- |
| 6 | Pcon-PT181 attenuator-aptamer12-1R-DT-10 | -- | -- |
| 7 | Pcon-PT181 attenuator-aptamer12-1R-DT-11 | -- | -- |
| 8 | Pcon-PT181 attenuator-aptamer12-1R-DT-12 | -- | -- |
| 9 | 1kbp ladder | -- | -- |
| 10 | Pcon-PT181 attenuator-RBS-GFP-DT-5 | -- | -- |
| 11 | Pcon-PT181 attenuator-RBS-GFP-DT-6 | -- | -- |
| 12 | Pcon-PT181 attenuator-RBS-GFP-DT-7 | -- | -- |
| 13 | Pcon-PT181 attenuator-RBS-GFP-DT-8 | -- | -- |
| 14 | Pcon-PT181 attenuator-pSB1C3-5 | -- | -- |
| 15 | Pcon-PT181 attenuator-pSB1C3-6 | -- | -- |
| 16 | Pcon-PT181 attenuator-pSB1C3-7 | -- | -- |
| 17 | Pcon-PT181 attenuator-pSB1C3-8 | -- | -- |
Transformation
| Name | Sample | Competent Cells | Total | Plate |
|---|---|---|---|---|
| Plac+tet aptamer12+Pcon tetR-DT+Ptet+RBS-GFP-DT | 1µL | 10µL | 11µL | - |
| Pcon-pT181attenuator-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT | 1µL | 10µL | 11µL | - |
| Pcon-tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT | 1µL | 10µL | 11µL | - |
| Pcon-Spinach-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT | 1µL | 10µL | 11µL | - |
| Pcon-pT181antisense+DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT | 1µL | 10µL | 11µL | - |
| Pcon-pT181attenuator+tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT | 1µL | 10µL | 11µL | - |
| Pcon tetR-DT+Ptet+RBS-GFP-DT | 1µL | 10µL | 11µL | - |
| Pcon tetR-DT+Pcon-GFP-DT+RBS-GFP-DT | 1µL | 10µL | 11µL | - |
| Pcon-tet aptamer12-DT+Pcon-pT181attenuator+RBS-GFP-DT | 1µL | 10µL | 11µL | - |
| Pcon-pT181attenuator-DT+Pcon-pT181attenuator+RBS-GFP-DT | 1µL | 10µL | 11µL | - |
| Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-RBS-GFP-DT+RBS-GFP-DT | 1µL | 10µL | 11µL | - |
| Pcon-tet aptamer12-DT+Pcon-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT | 1µL | 10µL | 11µL | - |
| Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT | 1µL | 10µL | 11µL | - |
| Name | Sample | Competent Cells | Total | Plate |
|---|---|---|---|---|
| aptamer12-DT(EcoRI+Xbal)+Pcon-pT181attenuator(EcoRI+Spel) | 2µL | 20µL | 22µL | - |
| Ptet(Spel+Pstl)+RBS-GFP-DT(Xbal+Pstl)) | 2µL | 20µL | 22µL | - |
Ligation
| state | Vector | Inserter | Ligation High ver.2 | ||
|---|---|---|---|---|---|
| experiment | 9/25 aptamer12_1R-DT(EcoRI&XbaI) | 4.5 µL | Pcon-pT181 attenuator(EcoRI&SpeI) | 7.7 µL | 6.1 µL |
| experiment | 9/17 pSB1C3(EcoRI&SpeI) | 2.0 µL | 9/3 pT181 attenuator(EcoRI&SpeI) | 14 µL | 8 µL |
| experiment | 9/25 Pcon-pT181 attenuator(EcoRI&XbaI) | 4.5 µL | 9/25 RBS-GFP-DT(EcoRI&SpeI) | 19.3 µL | 11.9 µL |
| experiment | 9/25 Pcon-RBS-GFP-DT(EcoRI&XbaI) | 4.2 µL | Pcon-RBS-tetR-DT(EcoRI&SpeI) | 20 µL | 12.1 µL |
Digestion
Liquid Culture
| Sample | medium |
|---|---|
| DT | - |
| pT181attenuator | - |
| Pcon-RBS-tetR-DT | - |
Gel Extraction
| Lane | DNA | Enzyme |
|---|---|---|
| （＞ω・）b | ||
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| （´ω｀） | |||
| （ΦωΦ^） |
Restriction Enzyme Digestion
| DNA | restricion enzyme | restriction enzyme | Buffer | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 2 cuts | （°°） µL | µL | µL | µL | µL | µL | µL |
| NC | µL | µL | µL | µL | µL | µL | µL |
Liquid Culture
| Sample | medium |
|---|---|
| (・ω・) | |
| （・Ｘ・） |
Ligation
| state | Vector | Inserter | Ligation High ver.2 | ||
|---|---|---|---|---|---|
| experiment | name | xx µL | name | xx µL | xx µL |
| NC | name | xx µL | name | xx µL | xx µL |
PCR
| genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | SasA_fwd primer | SasA_rev primer | MilliQ | total |
|---|---|---|---|---|---|---|---|---|
| 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 25 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| °C | °C | °C | °C | -- |
| （◎Д◎） |
Transformation
| Name | Sample | Competent Cells | Total | Plate |
|---|---|---|---|---|
| Plac+tet aptamer12+Pcon tetR-DT+Ptet+RBS-GFP-DT | 1µL | 10µL | 11µL | - |
| Pcon-pT181attenuator-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT | 1µL | 10µL | 11µL | - |
| いでんし | なんちゃらµL | ごにょごにょµL | ほにゃららµL | こーせーぶっしつ |
| いかどうよう | ||||
Electrophoresis
精製？
Restriction Enzyme Digestion
| DNA | restricion enzyme | restriction enzyme | Buffer | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 2 cuts | （°°） µL | µL | µL | µL | µL | µL | µL |
| NC | µL | µL | µL | µL | µL | µL | µL |
液体培養
プレーティング
Colony PCR
| Sample | base pair |
|---|---|
| (｀・ω・´) |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| °C | °C | °C | °C | -- |
| （◎Д◎） |
RNA抽出
cDNA合成
Electrophoresis
Ligation
| state | Vector | Inserter | Ligation High ver.2 | ||
|---|---|---|---|---|---|
| experiment | name | xx µL | name | xx µL | xx µL |
| NC | name | xx µL | name | xx µL | xx µL |
