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Team:Stockholm/16 October 2010
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==Andreas== | ==Andreas== | ||
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**pEX.nTAT⋅SOD⋅His | **pEX.nTAT⋅SOD⋅His | ||
**pEX.nLMWP⋅SOD⋅His | **pEX.nLMWP⋅SOD⋅His | ||
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| + | == Mimmi == | ||
| + | |||
| + | === clone non-CPP operon === | ||
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| + | (Andreas digested the operon from the vector pSB1C3 and ligated into pEX) | ||
| + | |||
| + | |||
| + | ==== Transformation ==== | ||
| + | |||
| + | * 66µl thawed cells + 2µl ligated vector | ||
| + | |||
| + | *Incubate on ice 30min | ||
| + | *Heat shock 55s in 42°C, cool down on ice | ||
| + | *Add 900µl LB, incubate in 37°C for 1h | ||
| + | *Spinn down cells and remove 900µl supernatant | ||
| + | *Resuspend cells and plate on LB<sub>amp</sub> + 0.1M IPTG (50µl) | ||
| + | |||
| + | {{Stockholm/Footer}} | ||
Latest revision as of 11:10, 26 October 2010
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Contents |
Andreas
Transfer of operon constructs into pEX
Continued Mimmi's experiments'
Plasmid prep
- 40 μl elution buffer (x2)
| DNA concentration | ||
|---|---|---|
| Sample | Conc [ng/μl] | A260/A280 |
| pSB1C3.SOD.yCCS | 188.2 | 1.94 |
| pSB1C3.SOD⋅His.yCCS | 223.6 | 1.91 |
| pSB1C3.His⋅SOD.yCCS | 230.6 | 1.93 |
Digestions
| pSB1C3. SOD.yCCS | pSB1C3. SOD⋅His.yCCS | pSB1C3. His⋅SOD.yCCS | |
|---|---|---|---|
| 10X FastDigest buffer | 2 | 2 | 2 |
| DNA (1 μg) | 5.3 | 4.5 | 4.3 |
| dH2O | 10.7 | 11.5 | 11.7 |
| FD XbaI | 1 | 1 | 1 |
| FD PstI | 1 | 1 | 1 |
| 20 μl | 20 μl | 20 μl |
- Incubation: 37 °C, 30 min
- Inactivation: 80 °C, 20 min
Ligations
Vector: pre-digested pEX.RFP (X+P)
| pEX.SOD. yCCS | pEX. SOD⋅His. yCCS | pEX. His⋅SOD. yCCS | |
|---|---|---|---|
| 10X T4 Ligase buffer | 2 | 2 | 2 |
| Vector DNA | 3 | 3 | 3 |
| Insert DNA | 9 | 9 | 9 |
| dH2O | 5 | 5 | 5 |
| T4 DNA ligase | 1 | 1 | 1 |
| 20 μl | 20 μl | 20 μl |
- Incubation: 22 °C, 15 min
Transformation
By Mimmi
nCPP culture growth assay
Prepared new clones to repeat the experiment from 15/10 by transforming new BL21.
Quick transformation protocol.
- 50 μl competent BL21 (30 μl plated)
- 0.5 μl plasmid DNA
- pEX.SOD⋅His
- pEX.nTra10⋅SOD⋅His
- pEX.nTAT⋅SOD⋅His
- pEX.nLMWP⋅SOD⋅His
Mimmi
clone non-CPP operon
(Andreas digested the operon from the vector pSB1C3 and ligated into pEX)
Transformation
- 66µl thawed cells + 2µl ligated vector
- Incubate on ice 30min
- Heat shock 55s in 42°C, cool down on ice
- Add 900µl LB, incubate in 37°C for 1h
- Spinn down cells and remove 900µl supernatant
- Resuspend cells and plate on LBamp + 0.1M IPTG (50µl)
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