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Team:Stockholm/30 September 2010
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Andreas
Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX
Digestions
- pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS
- Clones 2 & 3
- pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS
- Clones 1 & 2
- pSB1K3.nLMWP⋅SOD⋅His.RBS.yCCS
- Clones 2 & 3
| 1:2 | 1:3 | 2:1 | 2:2 | 3:2 | 3:3 | |
| 10X FastDigest buffer | 2 | 2 | 2 | 2 | 2 | 2 |
| DNA (1 μg) | 5.2 | 4.1 | 2.5 | 3.3 | 4 | 4.1 |
| dH2O | 10.8 | 11.9 | 13.5 | 12.7 | 12 | 11.9 |
| FD XbaI | 1 | 1 | 1 | 1 | 1 | 1 |
| FD PstI | 1 | 1 | 1 | 1 | 1 | 1 |
| 20 μl | 20 μl | 20 μl | 20 μl | 20 μl | 20 μl |
|---|
- Incubation: 37 °C, 1.45
- Inactivation: 80 °C, 20 min
Ligations
- Vector: [Dig pEX.RFP X+P
| pEX.1:2 | pEX.1:3 | pEX.2:1 | pEX.2:2 | pEX.3:2 | pEX.3:3 | |
| 10X T4 Ligase buffer | 2 | 2 | 2 | 2 | 2 | 2 |
| Vector DNA | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
| Insert DNA | 8 | 8 | 8 | 8 | 8 | 8 |
| dH2O | 7.5 | 7.5 | 7.5 | 7.5 | 7.5 | 7.5 |
| T4 DNA ligase | 1 | 1 | 1 | 1 | 1 | 1 |
| 20 μl | 20 μl | 20 μl | 20 μl | 20 μl | 20 μl |
|---|
- Incubation: 22 °C, 15 min
Transformation
Quick transformation
- 1 μl ligation mix
- 50 μl 0.1 M IPTG
- pEX.1:2 (pEX.nTAT⋅SOD⋅His.RBS.yCCS 2)
- pEX.1:3 (pEX.nTAT⋅SOD⋅His.RBS.yCCS 2)
- pEX.2:1 (pEX.nTra10⋅SOD⋅His.RBS.yCCS 1)
- pEX.2:2 (pEX.nTra10⋅SOD⋅His.RBS.yCCS 2)
- pEX.3:2 (pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2)
- pEX.3:3 (pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3)
Transformation of BL21
Quick transformation
- 50 μl competent cells
- 0.5 μl plasmid
- pEX.nTra10⋅SOD⋅His
- pEX.nLMWP⋅SOD⋅His
ON cultures
- 3 ml LB + appropriate antibiotic; 30 °C
- pEX.nTAT⋅SOD⋅His (Top10; Amp 100)
- pSB1K3.BBa_J04450 (Top10; Km 50)
