From 2013.igem.org
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- | ===Colony PCR===
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- | <div class="experiment">
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- | <span class="author">No name</span>
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- | {| class="wikitable"
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- | !Sample||base pair
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- | |-
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- | |pSB1C3+P-KaiBC①||283
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- | |-
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- | |pSB1C3+P-KaiBC②||283
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- | |}
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- | {| class="wikitable"
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- | !PreDenature||Denature||Annealing||Extension||cycle
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- | |-
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- | |94°C||94°C||55°C||68°C||--
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- | |-
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- | |2min||30s||30s||18s||30cycles
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- | |}
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| ===Ligation=== | | ===Ligation=== |
Revision as of 06:30, 26 September 2013
Sep 22
Colony PCR
Tatsui
Sample | base pair
|
9/20 Plac-PT181 attenuator(1~4) | 664
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9/20 Plac-PT181 antisense(1~6) | 369
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9/20 Pcon-PT181 attenuator-aptamer-DT(1~6) | 859
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9/20 Pcon-PT181 antisense-spinach-DT(1~8) | 723
|
Miniprep
Honda
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
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9/21 Plux-RBS-GFP-DT-(1) | 168.0 | 1.62 | 1.73
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9/21 Pcon-PT181 attenuator-(1) | 368.3 | 1.57 | 1.62
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9/21 RBS-GFP-DT | 532.8 | 1.75 | 1.78
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9/21 Pcon-RBS-GFP-DT | 845.2 | 2.02 | 1.70
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Colony PCR
Honda
Sample | base pair
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9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1~2) | 283
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PreDenature | Denature | Annealing | Extension | cycle
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94°C | 94°C | 55°C | 68°C | --
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5min | 30s | 30s | 2min10s | 30cycles
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No name
Lane | DNA | Enzyme
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1 | 100bp ladder | --
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2~4 | Pcon-aptamer12-1R-DT(E-1A) | --
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6~8 | Pcon-spinach-DT(E-1A) | --
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10~12 | Pcon-attenuator PT181(E-1A) | --
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Lane | DNA | Enzyme
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1 | 100bp ladder | --
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2~4 | DT(1-3) | --
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6~8 | aptamer12-1RDT(1-3) | --
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10~12 | Pcon-PT181 attenuator(1-5A) | --
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Name | concentration[µg/mL] | 260/280 | 260/230
|
Pcon-aptamer12-1R-DT(E-1A) | 31.2 | 1.87 | 0.30
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Pcon-spinach-DT(E-1A) | 25.6 | 1.88 | 0.99
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Pcon-attenuator PT181(E-1A) | 25.3 | 1.99 | 0.86
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DT(1-3) | 22.1 | 1.82 | 0.20
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aptamer12-1RDT(1-3) | 33.5 | 1.99 | 0.05
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Pcon-PT181 attenuator(1-5A) | 36.9 | 1.12 | 0.96
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digestion
No name
| 9/16 Plac | XbaI | SpeI | PstI | BufferB | BSA | MilliQ | total
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2cuts | 12.5µL | 0µL | 1µL | 1µL | 3µL | 3µL | 9.5µL | 30µL
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NC | 0.8µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.4µL | 10µL
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| 9/22 Plux | XbaI | SpeI | PstI | BufferB | BSA | MilliQ | total
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2cuts | 16µL | 0µL | 1µL | 1µL | 3µL | 3µL | 14µL | 30µL
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NC | 0.8µL | 0µL | | 0µL | 0µL | 1µL | 1µL | 7.2µL | 10µL
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| 9/20 RBS-tetR-2R-DT | XbaI | SpeI | PstI | BufferD | BSA | MilliQ | total
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2cuts | 8.5µL | 1µL | 0µL | 1µL | 3µL | 3µL | 13.5µL | 30µL
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NC | 0.4µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL
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No name
Lane | DNA | Enzyme
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1 | 100bp ladder | --
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2~4 | Pcon-attenuator PT181(E-0) | --
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Lane | DNA | Enzyme
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2 | 100bp ladder | --
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4~6 | Pcon-attenuator PT181(2-SA) | --
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Lane | DNA | Enzyme
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1 | 100bp ladder | --
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3~5 | spinach-DT(3-2) | --
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8~10 | Ptet-antisense PT181(1-3) | --
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Name | concentration[µg/mL] | 260/280 | 260/230
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Pcon-attenuator PT181(E-0) | 2.4 | 1.80 | 0.17
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Pcon-attenuator PT181(2-SA) | 10.8 | 1.85 | 0.54
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spinach-DT(3-2) | 36.7 | 1.80 | 0.75
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Ptet-antisense PT181(1-3) | 16.8 | 1.74 | 0.78
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Colony PCR
No name
Sample | base pair
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9/20 Pcon-PT181 attenuator-aptamer12-1R-DT-(7~14) | 859
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Colony PCR
Tatsui
Sample | base pair
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PconPT181antisense-spinach-DT(9~15) | 723
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Ligation
Okazaki, Kanaoka, Tatsui and Hirano
state | Vector | Inserter | Ligation High ver.2
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experiment | 9/18 Plac (SpeI & PstI) | 3.0 µL | 9/18 RBS-lysis3-DT (XbaI & PstI) | 12.4 µL | 3.5 µL
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experiment | 9/12 Ptet-pT181 antisense | 1.0 µL | 9/18 spinach-DT | 11.9 µL | 3.5 µL
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experiment | 9/13 Pcon-RBS-luxR-DT | 2.1 µL | 9/10 Plux-RBS-GFP-DT | 5.6 µL | 3.5 µL
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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
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experiment | 9/18 Plac ②(SpeI & PstI) | 3.0 µL | 9/18 aptamer 12-1R-DT(XbaI & PstI) | 4.5µL | x3.5 µL
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Liquid Culture
No name
Sample | medium
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9/2 spinach-DT-2 | --
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8/16 Plux-4 | --
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8/27 Plux-RBS-GFP-DT-1 | --
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9/5 Pcon-pT181 attenuator-1 | --
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digestion
Transformation
Tatsui
Name | Sample | Competent Cells | Plate
|
9/17 pSB1C3-Pcon-pT181 attenuator-DT | 2 µL | 20 µL | CP
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9/17 pSB1C3-Pcon-aptamer 12_1R-DT | 2 µL | 20 µL | CP
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9/17 pSB1C3-Pcon-antisense-spinach-DT | 2 µL | 20 µL | CP
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9/17 pSB1C3-Pcon-RBS-tetR-DT x2 | 2 µL | 20 µL | CP
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9/14 pSB1C3-PkaiBC | 2 µL | 20 µL | CP
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9/14 pSB1C3-RpaB | 2 µL | 20 µL | CP
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9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI) | 2 µL | 20 µL | CP
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9/17 pSB1C3-pT181 attenuator(XbaI&PstI) | 2 µL | 20 µL | CP
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9/17 pSB1C3-Pcon-spinach-DT | 2 µL | 20 µL | CP
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9/14 RBS-GFP-DT-PkaiBC | 2 µL | 20 µL | CP
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Plux-RBS-lysis3-DT | 2 µL | 20 µL | CP
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9/20 Ptet-pT181 antisense-spinach-DT | 2 µL | 20 µL | CP
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9/20 Pcon-spinach-DT(pSB4K5) | 2 µL | 20 µL | Kan
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9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5) | 2 µL | 20 µL | Kan
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9/20 KaiABC | 2 µL | 20 µL | Kan
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9/20 KaiABC | 2 µL | 20 µL | Kan
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9/20 Plac-RBS-lysis3-DT | 2 µL | 20 µL | Amp
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9/20 Plac-aptamer 12_1R | 2 µL | 20 µL | Amp
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9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT | 2 µL | 20 µL | Amp
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Plac-aptamer12_1R-DT | 2 µL | 20 µL | Amp
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9/20 Plac-aptamer12_1R-DT | 2 µL | 20 µL | Amp
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9/20Pcon-pT181attenuator-aptamer-DT | 2 µL | 20 µL | Amp
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9/17 Plac-pT181-attenuatorx2 | 2 µL | 20 µL | Amp
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9/15 Pcon-RBS-lacZα-DT | 2 µL | 20 µL | Amp
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9/14 Pcon-pT181attenuator-RBS-lacZα-DT | 2 µL | 20 µL | Amp
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PUC19 | 2 µL | 20 µL | Amp
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PCR
Tatsui
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
Tatsui
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
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9/20 Pcon-spinach-DT(pSB4K5) | 2 µL | 20 µL | Kan
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9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5) | 2 µL | 20 µL | Kan
|
9/20 KaiABC | 2 µL | 20 µL | Kan
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9/20 KaiABC | 2 µL | 20 µL | Kan
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9/20 Plac-RBS-lysis3-DT | 2 µL | 20 µL | Amp
|
9/20 Plac-aptamer 12_1R | 2 µL | 20 µL | Amp
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9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT | 2 µL | 20 µL | Amp
|
Plac-aptamer12_1R-DT | 2 µL | 20 µL | Amp
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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
|