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From 2013.igem.org

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;
  • 1. Resistance/Viability test
    2. Ligation experiment with 10ul DNA
    3. Ligation experiment with 5ul DNA
    4. 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