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Template:Kyoto/Notebook/Sep 17
From 2013.igem.org
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===Mutation PCR=== | ===Mutation PCR=== | ||
| - | |||
<div class="experiment"> | <div class="experiment"> | ||
<span class="author">Kinose</span> | <span class="author">Kinose</span> | ||
{| class="wikitable" | {| class="wikitable" | ||
| - | !pC KaiABC(100pg)||Prime STAR(2x)||Primer(KaiA- | + | !pC KaiABC(100pg/µL)||Prime STAR(2x)||Primer(KaiA-BsaI MuT-Fwd)||Primer(KaiA-BsaI MuT-Rev)||MilliQ||total |
|- | |- | ||
|1.0||25.0||1.0||1.0||22.0||50.0 | |1.0||25.0||1.0||1.0||22.0||50.0 | ||
| Line 211: | Line 210: | ||
!PreDenature||Denature||Annealing||Extension||cycle | !PreDenature||Denature||Annealing||Extension||cycle | ||
|- | |- | ||
| - | |96°C||98°C||65°C||72°C||-- | + | |96 °C||98 °C||65 °C||72 °C||-- |
|- | |- | ||
| - | | | + | |5 min||10 s||15 s||40 s||30 cycles |
|} | |} | ||
</div> | </div> | ||
Revision as of 13:54, 24 September 2013
Contents |
Sep 17
Liquid Culture
| Sample | medium |
|---|---|
| Spinach-DT | LB medium(+CP) |
| Pcon-Spinach-DT | LB medium(+Amp) |
| Pcon-pT181 antisense-Spinach-DT | LB medium(+Amp) |
PCR
| Pcon-tetR aptamer-DT(VF) | BigDye | 5x buffer | primer(VF) | MilliQ | total |
|---|---|---|---|---|---|
| 1.3 | 0.5 | 1.75 | 0.5 | 0.45 | 10.5 |
| Pcon-tetR aptamer-DT(VR) | BigDye | 5x buffer | primer(VR) | MilliQ | total |
|---|---|---|---|---|---|
| 1.3 | 0.5 | 1.75 | 0.5 | 0.45 | 10.5 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 96 °C | 96 °C | 50 °C | 60 °C | -- |
| 2 min | 10 sec | 5 sec | 4 min | 30 cycles |
Electrophoresis
| Lane | Sample | Enzyme1 | Enzyme2 |
|---|---|---|---|
| 1 | 1kbp ladder | -- | -- |
| 2 | Pcon-RBS-tetR-DT | XbaI | PstI |
| 3 | Pcon-RBS-tetR-DT | -- | -- |
| 4 | pSB4K5 | XbaI | PstI |
| 5 | pSB4K5 | -- | -- |
| 6 | pSB1C3 | EcoRI | SpeI |
| 7 | pSB1C3 | EcoRI | PstI |
| 8 | pSB1C3 | -- | -- |
| 9 | pT181 attenuator | EcoRI | SpeI |
| 10 | pT181 attenuator | -- | -- |
| 11 | 1kbp ladder | -- | -- |
Ligation
| state | Vector | Inserter | Ligation High ver.2 | ||
|---|---|---|---|---|---|
| experiment | 8/28 Plux (SpeI & PstI) | 5.0 µL | 9/13 RBS-lysis1-DT (XbaI & PstI) | 3.5 µL | 3.5 µL |
| experiment | 8/28 Plux (SpeI & PstI) | 5.0 µL | 9/13 RBS-lysis2-DT (XbaI & PstI) | 3.6 µL | 3.5 µL |
| experiment | 8/28 Plux (SpeI & PstI) | 11.0 µL | 9/13 RBS-lysis3-DT (XbaI & PstI) | 9.5 µL | 3.5 µL |
| experiment | 9/14 Plac (SpeI & PstI) | 2.4 µL | 9/16 pT181 attenuator-DT (XbaI & PstI) | 4.8 µL | 3.5 µL |
| experiment | 9/14 Plac (SpeI & PstI) | 2.4 µL | 9/3 pT181 antisense (XbaI & PstI) | 2.0 µL | 2.2 µL |
| experiment | 9/14 Plac (SpeI & PstI) | 2.4 µL | 9/10 aptamer 12_1R-DT (XbaI & PstI) | 1.8 µL | 2.1 µL |
| experiment | 9/13 pSB1C3 (XbaI & PstI) | 2.0 µL | 9/15 Pcon-PT181 antisense-Spinach-DT (XbaI & PstI) | 12.0 µL | 3.5 µL |
| experiment | 9/16 pSB1C3 (EcoRI & SpeI) | 1.0 µL | 9/15 Pcon-Spinach-DT (EcoRI & SpeI) | 4.1 µL | 2.0 µL |
| experiment | 9/16 pSB1C3 (EcoRI & SpeI) | 1.0 µL | 9/15 Pcon-RBS-tetR-DT (EcoRI & SpeI) | 2.0 µL | 1.5 µL |
| experiment | 9/16 pSB1C3 (EcoRI & SpeI) | 1.0 µL | 9/15 Pcon-PT181 attenuator-DT (EcoRI & SpeI) | 2.4 µL | 1.7 µL |
| experiment | 9/16 pSB1C3 (EcoRI & SpeI) | 1.0 µL | 9/15 Pcon-aptamer 12_1R-DT (EcoRI & SpeI) | 17.0 µL | 1.5 µL |
| experiment | 9/16 pSB1C3 (XbaI & PstI) | 1.0 µL | 9/15 Pcon-aptamer 12_1R-DT (EcoRI & SpeI) | 4.4 µL | 3.5 µL |
Gel Extraction
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 1kbp ladder | -- |
| 2 | pT181 attenuator -(2) | EcoRI & SpeI |
| 3 | ||
| 4 |
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| pT181 attenuator (EcoI & SpeI) | 9.2 | 1.15 | 0.12 |
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 1kbp ladder | -- |
| 2 | pSB1C3 | EcoRI & SpeI |
| 3 | ||
| 4 |
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| pSB1C3 | 10.0 | 1.84 | 0.40 |
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 1kbp ladder | -- |
| 2 | pSB4K5 | XbaI & PstI |
| 3 | ||
| 4 |
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| pSB4K5 | 7.6 | 1.81 | 0.32 |
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 1kbp ladder | -- |
| 2 | Pcon-RBS-tetR-DT | XbaI & PstI |
| 3 | ||
| 4 |
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| Pcon-RBS-tetR-DT | 8.0 | 2.40 | 1.14 |
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 1kbp ladder | -- |
| 2 | pSB1C3 | EcoRI & PstI |
| 3 | ||
| 4 |
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| pSB1C3 | 9.1 | 2.17 | 0.35 |
Mutation PCR
| pC KaiABC(100pg/µL) | Prime STAR(2x) | Primer(KaiA-BsaI MuT-Fwd) | Primer(KaiA-BsaI MuT-Rev) | MilliQ | total |
|---|---|---|---|---|---|
| 1.0 | 25.0 | 1.0 | 1.0 | 22.0 | 50.0 |
| 1.5 | 25.0 | 1.0 | 1.0 | 21.5 | 50.0 |
| 2.0 | 25.0 | 1.0 | 1.0 | 21.0 | 50.0 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 96 °C | 98 °C | 65 °C | 72 °C | -- |
| 5 min | 10 s | 15 s | 40 s | 30 cycles |
