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Template:Kyoto/Notebook/Sep 6
From 2013.igem.org
Contents |
Sep 6
Colony PCR
| Sample | base pair |
|---|---|
| Pconst-spinach-DT-(1~4) | 563 |
| Plac-spinach-DT-(1~4) | 659 |
| Pcon-PT181 antisense-(1~4) | 468 |
| Ptet-PT181 antisense-(1~4) | 604 |
| Pconst-PT181 attenuater-(1~4) | 568 |
| Pconst＋RBS-tetR-DT-(1~4) | 1121 |
| Ptet＋RBS-lacZα-DT-(1~4) | 1162 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 40s | 30cycles |
Liquid Culture
| Sample | medium |
|---|---|
| 9/4 RBS-Lysis3＋DT3 | Plusgrow medium(+Cp) |
| 9/4 RBS-Lysis1＋DT3 | Plusgrow medium(+Cp) |
| 9/4 PSB4K5 | Plusgrow medium(+K) |
Colony PCR
| Sample | base pair |
|---|---|
| Pconst-spinach-DT-(1) | - |
| Pconst-spinach-DT-(2) | - |
| Pconst-spinach-DT-(3) | - |
| Pconst-spinach-DT-(4) | - |
| Plac-spinach-DT-(1) | - |
| Plac-spinach-DT-(2) | - |
| Plac-spinach-DT-(3) | - |
| Plac-spinach-DT-(4) | - |
| Pcon-PT181 antisense-(1) | - |
| Pcon-PT181 antisense-(2) | - |
| Pcon-PT181 antisense-(3) | - |
| Pcon-PT181 antisense-(4) | - |
| Sample | base pair |
|---|---|
| Ptet-PT181 antisense-(1) | - |
| Ptet-PT181 antisense-(2) | - |
| Ptet-PT181 antisense-(3) | - |
| Ptet-PT181 antisense-(4) | - |
| Plac-PT181 attenuater-(1) | - |
| Plac-PT181 attenuater-(2) | - |
| Plac-PT181 attenuater-(3) | - |
| Plac-PT181 attenuater-(4) | - |
Miniprep
| DNA | concentration [µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| PT181 antisense-(1) | 238.7 | 1.68 | 1.44 |
| PT181 antisense-(2) | 248.8 | 1.83 | 1.77 |
| PT181 antisense-(3) | 223.2 | 1.78 | 1.61 |
| PT181 antisense-(4) | 220.8 | 1.89 | 1.92 |
| tetR aptamer-(1) | 252.5 | 1.89 | 1.90 |
| tetR aptamer-(2) | 211.2 | 1.90 | 1.99 |
| DT181 attenuater | 102.2 | 1.98 | 2.17 |
| 121R aptamer-DT | 251.7 | 1.92 | 2.01 |
| PSB4K5 | 212.9 | 1.80 | 1.90 |
| RBS-lacZα-DT | 185.7 | 1.78 | 1.79 |
| RBS-lysis2-DT-(1) | 84.8 | 1.85 | 2.24 |
Restriction Enzyme Digestion
| 9/6 RBS-lysis2-DT (78.3 µg/mL) | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
|---|---|---|---|---|---|---|---|---|---|
| 2 cuts | 22µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 0µL | 30µL |
| NC | 1.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 6.7µL | 10µL |
- at 37°C, for 1h
| aptamer-DT (251.7 µg/mL) | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
|---|---|---|---|---|---|---|---|---|---|
| 2 cuts | 0.4µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 14.1µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |
- at 37°C, for 1h
| Pλ-luxI (86.4 µg/mL) | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
|---|---|---|---|---|---|---|---|---|---|
| 2 cuts | 23.1µL | 1µL | 1µL | 0µL | 0µL | 3µL | 3µL | 1.9µL | 30µL |
| NC | 1.2µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 6.8µL | 10µL |
- at 37°C, for 1h
Colony PCR
| Sample | base pair |
|---|---|
| Pconst＋RBS-tetR-DT-(1) | 1121 |
| Pconst＋RBS-tetR-DT-(2) | 1121 |
| Pconst＋RBS-tetR-DT-(3) | 1121 |
| Pconst＋RBS-tetR-DT-(4) | 1121 |
| Ptet＋RBS-lacZα-DT-(1) | 1162 |
| Ptet＋RBS-lacZα-DT-(2) | 1162 |
| Ptet＋RBS-lacZα-DT-(3) | 1162 |
| Ptet＋RBS-lacZα-DT-(4) | 1162 |
Gel Extraction
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 100bp ladder | -- |
| 2 | RBS-lysis2-DT | XbaI & PstI |
| 3 | ||
| 4 | -- | -- |
| 5 | aptamer 12-1R-DT | XbaI & PstI |
| 6 |
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 100bp ladder | -- |
| 2 | Pλ-lux3 | EcoRI & SpeI |
| 3 | ||
| 4 | ||
| 5 | ||
| 6 |
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| RBS-lysis2-DT (XbaI&PstI) | 7.1 | 1.88 | 0.07 |
| aptamer 12-1R-DT (XbaI&PstI) | 15.3 | 1.93 | 0.70 |
| Pλ-lux3 (EcoRI&SpeI) | 4.5 | 1.79 | 0.31 |
Liquid Culture
| Sample | medium |
|---|---|
| 9/5 Pconst-spinach-DT-(1) | Plusgrow medium(+Amp&CP) |
| 9/5 Ptet-PT181 antisense-(1) | Plusgrow medium(+CP) |
| 9/5 Pconst＋RBS-tetR-DT-(1) | Plusgrow medium(+Amp&CP) |
| 9/5 Ptet＋RBS-lacZα-DT-(1) | Plusgrow medium(+CP) |
| 9/5 Pcon-PT181 attenuater-(1) | Plusgrow medium(+CP) |
| 9/4 pT181 antisense-(1) | Plusgrow medium(+CP) |
| 9/4 aptamer 12-1R-(1) | Plusgrow medium(+CP) |
Colony PCR
| Sample | base pair |
|---|---|
| 9/5 Plac-spinach-DT-(5~12) | 563 |
| 9/5 Pcon-antisense-(5~12) | 468 |
| 9/5 Plac-attenuater-(5~12) | 664 |
| 9/5 PT181 antisense＋Plac-(1) | - |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 42s | 30cycles |
