From 2013.igem.org
Sep 9
PCR
Nakagawa
| KaiA(plasmid) | 10x buffer | dNTPs | MgSO4 | Primer(661-RBS-KaiA-fwd) | Primer(kaiA-662-rev-ver2) | Kod-plus ver2 | MilliQ | total
|
| 1.0 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.5 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 58.5°C | 68°C | --
|
| 2min | 10s | 30s | 30s | 30cycles
|
| KaiB(plasmid) | 10x buffer | dNTPs | MgSO4 | Primer(661-RBS-KaiA-fwd-ver2) | Primer(kaiB-663-rev-ver2) | Kod-plus ver2 | MilliQ | total
|
| 1.0 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.5 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 58.5°C | 68°C | --
|
| 2min | 10s | 30s | 30s | 30cycles
|
| P-KaiBC(PCR products) | 10x buffer | dNTPs | MgSO4 | Primer(prefix-P-kaiBC-fwd-ver2) | Primer(P-KaiBC-suffix-rev-verw) | Kod-plus ver2 | MilliQ | total
|
| 0.7 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.8 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 58.5°C | 68°C | --
|
| 2min | 10s | 30s | 30s | 30cycles
|
| DT(plasmid) | 10x buffer | dNTPs | MgSO4 | Primer(664-DT-fwd-ver2) | Primer(DT-suffix-660-rev-ver2) | Kod-plus ver2 | MilliQ | total
|
| 1.0 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.5 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 58.5°C | 68°C | --
|
| 2min | 10s | 30s | 30s | 30cycles
|
Miniprep
No name
| DNA | concentration[µg/mL] | 260/280 | 260/230
|
| 9/8 pSB1C3 | 83.2 | 1.45 | 0.76
|
| 9/8 RBS-lysis2-DT | 72.8 | 1.67 | 0.97
|
| 9/8 spinach-DT | 152.6 | 1.88 | 1.97
|
| Nuclease-Free water | -0.4 | -14.30 | 0.25
|
Restriction Enzyme Digestion
No name
| | 9/9 pSB1C3 | XbaI | PstI | Buffer | BSA | MilliQ | total
|
| 2 cuts | 24µL | 1µL | 1µL | 3µL | 3µL | (22)µL | 30µL
|
| NC | µL | µL | µL | µL | µL | µL | µL
|
No name
| | 9/9 pSB1C3 | XbaI | PstI | Buffer | BSA | MilliQ | total
|
| 2 cuts | 24µL | 1µL | 1µL | 3µL | 3µL | (22)µL | 30µL
|
| NC | 1.2µL | 0µL | 0µL | µL | µL | µL | µL
|
No name
| | 9/9 spinach-DT | XbaI | PstI | Buffer | BSA | MilliQ | total
|
| 2 cuts | 27.4µL | 1µL | 1µL | 3µL | 3µL | (22)µL | 30µL
|
| NC | µL | µL | µL | µL | µL | µL | µL
|
Transformation
じっけんしゃ
| Name | Sample | Competent Cells | Total | Plate
|
| 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 | | | |
|
| | | | |
|
Ligation
No Name
| state | Vector | Inserter | Ligation High ver.2
|
| experiment | 9/8 F1-attenuator(XbaI&PstI) | 5.3µg | 9/8 pSB1C3(XbaI&PstI) | 8.9µg | 3.5µg
|
| experiment | 9/8 F3m2-attenuator(XbaI&PstI) | 4.5µg | 9/8 pSB1C3(XbaI&PstI) | 8.9µg | 3.5µg
|
| experiment | 9/8 F1-antisense(XbaI&PstI) | 1.7µg | 9/8 pSB1C3(XbaI&PstI) | 8.9µg | 3.5µg
|
| experiment | 9/8 F6-antisense(XbaI&PstI) | 2.9µg | 9/8 pSB1C3(XbaI&PstI) | 8.9µg | 3.5µg
|
| experiment | 9/8 aptamer12-P(EcoRI&SpeI) | 3.1µg | 9/8 pSB1C3(EcoRI&SpeI) | 8.8µg | 3.5µg
|
| experiment | 9/8 aptamer12-1M(EcoRI&SpeI) | 3.1µg | 9/8 pSB1C(EcoRI&SpeI)3 | 8.8µg | 3.5µg
|
Fe(Ⅲ)
Shimizu & Hirano
Colony PCR
No name
| Sample | base pair
|
| 9/8 Plac-spinach-DT-(1) | 659
|
| 9/8 Plac-spinach-DT-(2) | 659
|
| 9/8 Pcon-pt181atte-DT-(1) | 584
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 5m | 30s | 30s | 40s | 30cycles
|
Ligation
じっけんしゃ
| state | Vector | Inserter | Ligation High ver.2
|
| experiment | name | xx µg/mL | name | xx µg/mL | xx µg/mL
|
| NC | name | xx µg/mL | name | xx µg/mL | xx µg/mL
|
PCR
Tatsui and Yoshida
| KaiC | 10x buffer KOD plus ver2 | 2M dNTP | MgSO4 | Primer Mix(F&R) | Kod-plus ver2 | MilliQ | total
|
| 0.5 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 16.0 | 25.0
|
| 1.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 15.5 | 25.0
|
| 2.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14.5 | 25.0
|
| 4.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 12.5 | 25.0
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 57°C | 68°C | --
|
| 2m | 10s | 30s | 2m | 30cycles
|
| Plac(100pg) | 10x buffer KOD plus ver2 | 2M dNTP | MgSO4 | Primer Mix(F&R) | Kod-plus ver2 | MilliQ | total
|
| 0.3 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 16.2 | 25.0
|
| 0.6 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 15.9 | 25.0
|
| 1.2 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 15.3 | 25.0
|
| 2.4 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14.1 | 25.0
|
| 0.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 16.5 | 25.0
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 57°C | 68°C | --
|
| 2m | 10s | 30s | 2m | 30cycles
|
Liquid Culture
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
|
PCR
Yoshida
| KaiA(KaiABC) 100pg/µL | 10x buffer KOD plus ver2 | dNTPs | MgSO4 | Primer Mix(Fwd&Rev) | KOD plus ver2 | MilliQ | total
|
| 2.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14.5 | 25
|
| 4.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 12.5 | 25
|
| 8.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 8.5 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 60°C | 68°C | --
|
| 2m | 10s | 30s | 1m | 30cycles
|
| PKaiBC 400pg/µL | 10x buffer KOD plus ver2 | dNTPs | MgSO4 | Primer Mix(Fwd&Rev) | KOD plus ver2 | MilliQ | total
|
| 0.5 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 16.0 | 25
|
| 1.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 15.5 | 25
|
| 2.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14.5 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 60°C | 68°C | --
|
| 2m | 10s | 30s | 1m | 30cycles
|
Liquid Culture
No name
| Sample | medium
|
| 9/2 Spinach-DT | Plusgrow medium(+CP)
|
| 8/29 Plux-RBS-GFP-DT | Plusgrow medium
|
| 9/6 master plate | Plusgrow medium(+Amp)
|
| 3 Pcon-PT181 attenuator | Plusgrow medium
|
Electrophoresis
Yoshida
| Lane | Sample | Enzyme1 | Enzyme2
|
| 1 | 1kb ladder | -- | --
|
| 2 | Plac 100pg | -- | --
|
| 3 | Plac 200pg | -- | --
|
| 4 | Plac 400pg | -- | --
|
| 5 | Plac 800pg | -- | --
|
| 6 | NC | -- | --
|
| 7 | 1kb ladder | -- | --
|
File:Igku xxxxxx.xxx
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