Template:Kyoto/Notebook/Sep 22
From 2013.igem.org
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===digestion=== | ===digestion=== | ||
+ | <div class="experiment"> | ||
+ | <span class="author">No name</span> | ||
+ | {| class="wikitable" | ||
+ | ! ||9/16 Plac||XbaI||SpeI||PstI||BufferB||BSA||MilliQ||total | ||
+ | |- | ||
+ | |2cuts||12.5µL||0µL||1µL||1µL||3µL||3µL||9.5µL||30µL | ||
+ | |- | ||
+ | |NC||0.8µL||0µL||0µL||0µL||1µL||1µL||7.4µL||10µL | ||
+ | |- | ||
+ | |} | ||
+ | {| class="wikitable" | ||
+ | ! ||9/22 Plux||XbaI||SpeI||PstI||BufferB||BSA||MilliQ||total | ||
+ | |- | ||
+ | |2cuts||16µL||0µL||1µL||1µL||3µL||3µL||14µL||30µL | ||
+ | |- | ||
+ | |NC||0.8µL||0µL||||0µL||0µL||1µL||1µL||7.2µL||10µL | ||
+ | |} | ||
+ | |||
+ | {| class="wikitable" | ||
+ | ! ||9/20 RBS-tetR-2R-DT ||XbaI||SpeI||PstI||BufferD||BSA||MilliQ||total | ||
+ | |- | ||
+ | |2cuts||8.5µL||1µL||0µL||1µL||3µL||3µL||13.5µL||30µL | ||
+ | |- | ||
+ | |NC||0.4µL||0µL||0µL||0µL||1µL||1µL||7.6µL||10µL | ||
+ | |- | ||
+ | |} | ||
===Gel Extraction=== | ===Gel Extraction=== |
Revision as of 06:00, 26 September 2013
Contents |
Sep 22
Colony PCR
Sample | base pair |
---|---|
9/20 Plac-PT181 attenuator(1~4) | 664 |
9/20 Plac-PT181 antisense(1~6) | 369 |
9/20 Pcon-PT181 attenuator-aptamer-DT(1~6) | 859 |
9/20 Pcon-PT181 antisense-spinach-DT(1~8) | 723 |
Miniprep
DNA | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
9/21 spinach-DT | 197.9 | 1.67 | 1.54 |
9/21 Plux | 125.9 | 1.71 | 1.27 |
9/21 Plux-RBS-GFP-DT-(1) | 168.0 | 1.62 | 1.73 |
9/21 Pcon-PT181 attenuator-(1) | 368.3 | 1.57 | 1.62 |
9/21 RBS-GFP-DT | 532.8 | 1.75 | 1.78 |
9/21 Pcon-RBS-GFP-DT | 845.2 | 2.02 | 1.70 |
Colony PCR
Sample | base pair |
---|---|
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1~2) | 283 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | -- |
5min | 30s | 30s | 2min10s | 30cycles |
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | -- |
2~4 | Pcon-pz12-1R-DT(E-1A) | -- |
6~8 | Pcon-spinach-DT(E-1A) | -- |
10~12 | Pcon-attenuator PT181(E-1A) | -- |
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | -- |
2~4 | DT(1-3) | -- |
6~8 | aptamer12-1RDT(1-3) | -- |
10~12 | Pcon-PT181 attenuator(1-5A) | -- |
digestion
9/16 Plac | XbaI | SpeI | PstI | BufferB | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|
2cuts | 12.5µL | 0µL | 1µL | 1µL | 3µL | 3µL | 9.5µL | 30µL |
NC | 0.8µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.4µL | 10µL |
9/22 Plux | XbaI | SpeI | PstI | BufferB | BSA | MilliQ | total | ||
---|---|---|---|---|---|---|---|---|---|
2cuts | 16µL | 0µL | 1µL | 1µL | 3µL | 3µL | 14µL | 30µL | |
NC | 0.8µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.2µL | 10µL |
9/20 RBS-tetR-2R-DT | XbaI | SpeI | PstI | BufferD | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|
2cuts | 8.5µL | 1µL | 0µL | 1µL | 3µL | 3µL | 13.5µL | 30µL |
NC | 0.4µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | -- |
3 | RBS-lacZα-DT | XbaI&PstI |
4 | RBS-lacZα-DT | XbaI&PstI |
5 | RBS-lacZα-DT | XbaI&PstI |
6 | RBS-lacZα-DT | XbaI&PstI |
File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
RBS-lacZα-DT(XbaI&PstI) | 9.8 | 1.50 | 0.39 |
Colony PCR
Sample | base pair |
---|---|
pSB1C3+P-KaiBC① | 283 |
pSB1C3+P-KaiBC② | 283 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | -- |
2min | 30s | 30s | 18s | 30cycles |
Colony PCR
Sample | base pair |
---|---|
pSB1C3+P-KaiBC① | 283 |
pSB1C3+P-KaiBC② | 283 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | -- |
2min | 30s | 30s | 18s | 30cycles |
Colony PCR
Sample | base pair |
---|---|
pSB1C3+P-KaiBC① | 283 |
pSB1C3+P-KaiBC② | 283 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | -- |
2min | 30s | 30s | 18s | 30cycles |
Ligation
state | Vector | Inserter | Ligation High ver.2 | ||
---|---|---|---|---|---|
experiment | 9/18 Plac (SpeI & PstI) | 3.0 µL | 9/18 RBS-lysis3-DT (XbaI & PstI) | 12.4 µL | 3.5 µL |
experiment | 9/12 Ptet-pT181 antisense | 1.0 µL | 9/18 spinach-DT | 11.9 µL | 3.5 µL |
experiment | 9/13 Pcon-RBS-luxR-DT | 2.1 µL | 9/10 Plux-RBS-GFP-DT | 5.6 µL | 3.5 µL |
experiment | 9/13 Pcon-pT181 attenuator (SpeI & PstI) | 2.0 µL | 9/18 aptamer 12-1R-DT (XbaI & PstI) | 6.3 µL | 3.5 µL |
experiment | 9/18 Plac ②(SpeI & PstI) | 3.0 µL | 9/18 aptamer 12-1R-DT(XbaI & PstI) | 4.5µL | x3.5 µL |
Liquid Culture
Sample | medium |
---|---|
9/2 spinach-DT-2 | -- |
8/16 Plux-4 | -- |
8/27 Plux-RBS-GFP-DT-1 | -- |
9/5 Pcon-pT181 attenuator-1 | -- |
digestion
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | -- |
3 | RBS-lacZα-DT | XbaI&PstI |
4 | RBS-lacZα-DT | XbaI&PstI |
5 | RBS-lacZα-DT | XbaI&PstI |
6 | RBS-lacZα-DT | XbaI&PstI |
File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
RBS-lacZα-DT(XbaI&PstI) | 9.8 | 1.50 | 0.39 |
Transformation
Name | Sample | Competent Cells | Plate |
---|---|---|---|
9/17 pSB1C3-Pcon-pT181 attenuator-DT | 2 µL | 20 µL | CP |
9/17 pSB1C3-Pcon-aptamer 12_1R-DT | 2 µL | 20 µL | CP |
9/17 pSB1C3-Pcon-antisense-spinach-DT | 2 µL | 20 µL | CP |
9/17 pSB1C3-Pcon-RBS-tetR-DT x2 | 2 µL | 20 µL | CP |
9/14 pSB1C3-PkaiBC | 2 µL | 20 µL | CP |
9/14 pSB1C3-RpaB | 2 µL | 20 µL | CP |
9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI) | 2 µL | 20 µL | CP |
9/17 pSB1C3-pT181 attenuator(XbaI&PstI) | 2 µL | 20 µL | CP |
9/17 pSB1C3-Pcon-spinach-DT | 2 µL | 20 µL | CP |
9/14 RBS-GFP-DT-PkaiBC | 2 µL | 20 µL | CP |
Plux-RBS-lysis3-DT | 2 µL | 20 µL | CP |
9/20 Ptet-pT181 antisense-spinach-DT | 2 µL | 20 µL | CP |
9/20 Pcon-spinach-DT(pSB4K5) | 2 µL | 20 µL | Kan |
9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5) | 2 µL | 20 µL | Kan |
9/20 KaiABC | 2 µL | 20 µL | Kan |
9/20 KaiABC | 2 µL | 20 µL | Kan |
9/20 Plac-RBS-lysis3-DT | 2 µL | 20 µL | Amp |
9/20 Plac-aptamer 12_1R | 2 µL | 20 µL | Amp |
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT | 2 µL | 20 µL | Amp |
Plac-aptamer12_1R-DT | 2 µL | 20 µL | Amp |
9/20 Plac-aptamer12_1R-DT | 2 µL | 20 µL | Amp |
9/20Pcon-pT181attenuator-aptamer-DT | 2 µL | 20 µL | Amp |
9/17 Plac-pT181-attenuatorx2 | 2 µL | 20 µL | Amp |
9/15 Pcon-RBS-lacZα-DT | 2 µL | 20 µL | Amp |
9/14 Pcon-pT181attenuator-RBS-lacZα-DT | 2 µL | 20 µL | Amp |
PUC19 | 2 µL | 20 µL | Amp |
PCR
Sample | base pair |
---|---|
pT181 attenuator(pSB1C3) | -- |
Pcon-RBS-lacZα-DT | -- |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94 °C | 94 °C | 55 °C | 68 °C | -- |
5 min | 30 s | 30 s | 3 min | 30 cycles |
Transformation
Name | Sample | Competent Cells | Plate |
---|---|---|---|
9/17 pSB1C3-Pcon-pT181 attenuator-DT | 2 µL | 20 µL | CP |
9/17 pSB1C3-Pcon-aptamer 12_1R-DT | 2 µL | 20 µL | CP |
9/17 pSB1C3-Pcon-antisense-spinach-DT | 2 µL | 20 µL | CP |
9/17 pSB1C3-Pcon-RBS-tetR-DT x2 | 2 µL | 20 µL | CP |
9/14 pSB1C3-PkaiBC | 2 µL | 20 µL | CP |
9/14 pSB1C3-RpaB | 2 µL | 20 µL | CP |
9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI) | 2 µL | 20 µL | CP |
9/17 pSB1C3-pT181 attenuator(XbaI&PstI) | 2 µL | 20 µL | CP |
9/17 pSB1C3-Pcon-spinach-DT | 2 µL | 20 µL | CP |
9/14 RBS-GFP-DT-PkaiBC | 2 µL | 20 µL | CP |
Plux-RBS-lysis3-DT | 2 µL | 20 µL | CP |
9/20 Ptet-pT181 antisense-spinach-DT | 2 µL | 20 µL | CP |
9/20 Pcon-spinach-DT(pSB4K5) | 2 µL | 20 µL | Kan |
9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5) | 2 µL | 20 µL | Kan |
9/20 KaiABC | 2 µL | 20 µL | Kan |
9/20 KaiABC | 2 µL | 20 µL | Kan |
9/20 Plac-RBS-lysis3-DT | 2 µL | 20 µL | Amp |
9/20 Plac-aptamer 12_1R | 2 µL | 20 µL | Amp |
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT | 2 µL | 20 µL | Amp |
Plac-aptamer12_1R-DT | 2 µL | 20 µL | Amp |
9/20 Plac-aptamer12_1R-DT | 2 µL | 20 µL | Amp |
9/20Pcon-pT181attenuator-aptamer-DT | 2 µL | 20 µL | Amp |
9/17 Plac-pT181-attenuatorx2 | 2 µL | 20 µL | Amp |
9/15 Pcon-RBS-lacZα-DT | 2 µL | 20 µL | Amp |
9/14 Pcon-pT181attenuator-RBS-lacZα-DT | 2 µL | 20 µL | Amp |
PUC19 | 2 µL | 20 µL | Amp |