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30/07/13
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==Transformation of the limonene Biobrick (BBa_K118025)== | ==Transformation of the limonene Biobrick (BBa_K118025)== | ||
Ligated with the pSB1C3 vector (restriction sites EcoRI, PstI) | Ligated with the pSB1C3 vector (restriction sites EcoRI, PstI) | ||
| + | |||
| + | Protocol | ||
| + | *Start thawing the competent cells on ice | ||
| + | *Seperate 4 pre-chilled eppendorf tubes; | ||
| + | **#Resistance/Viability test | ||
| + | **#Ligation experiment with 10ul DNA | ||
| + | **#Ligation experiment with 5ul DNA | ||
| + | **#Positive control using 1ul RFP | ||
| + | *Add 50ul of thawed competent cells and then the aforementioned volume of DNA to each eppendorf tube. | ||
| + | *For each eppendorf tube, pipette the solution gently to mix. Ensure the cells are kept on ice. | ||
| + | *Close the tubes and incubate the cells on ice for 30 minutes | ||
| + | *Heat shock the cells by immersion in a pre-heated water bath at 42'C for 60 seconds | ||
| + | *Incubate the cells on ice for 5 minutes | ||
| + | *Add the 200ul of SOC (SOB + 0.4g glucose) media to each tube | ||
| + | *Incubate the cells at 37'C for 2 hours | ||
Revision as of 11:06, 30 July 2013
Transformation of the limonene Biobrick (BBa_K118025)
Ligated with the pSB1C3 vector (restriction sites EcoRI, PstI)
Protocol
- Start thawing the competent cells on ice
- Seperate 4 pre-chilled eppendorf tubes;
- Resistance/Viability test
- Ligation experiment with 10ul DNA
- Ligation experiment with 5ul DNA
- Positive control using 1ul RFP
- Add 50ul of thawed competent cells and then the aforementioned volume of DNA to each eppendorf tube.
- For each eppendorf tube, pipette the solution gently to mix. Ensure the cells are kept on ice.
- Close the tubes and incubate the cells on ice for 30 minutes
- Heat shock the cells by immersion in a pre-heated water bath at 42'C for 60 seconds
- Incubate the cells on ice for 5 minutes
- Add the 200ul of SOC (SOB + 0.4g glucose) media to each tube
- Incubate the cells at 37'C for 2 hours
