From 2013.igem.org
Sep 25
Colony PCR
Nakamoto
| 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
no name
| 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 | -- | --
|
File:Xxxxx1.jpg
Transformation
kojima and nakamoto
| 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
nakamoto
| 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
Kojima
| Sample | medium
|
| DT | -
|
| pT181attenuator | -
|
| Pcon-RBS-tetR-DT | -
|
Restriction Enzyme Digestion
じっけんしゃ
| | 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
Ligation
じっけんしゃ
| 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
No name
| 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
じっけんしゃ
| 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
じっけんしゃ
| | 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
No name
| PreDenature | Denature | Annealing | Extension | cycle
|
| °C | °C | °C | °C | --
|
| （◎Д◎） | | | |
|
RNA抽出
cDNA合成
Electrophoresis
Ligation
じっけんしゃ
| 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
|
"
