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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
• Plate onto petri dishes (ensure your petri dishes are labelled correctly):
  • 1. For eppendorf 1, plate 100ul onto a chloroamphenicol petri dish and another 100ul of the solution onto LB (luria broth)
    2. For eppendorf 2, plate 200ul and 20ul onto two separate chloroamphenicol petri dishes
    3. For eppendorf 3, plate 200ul and 20ul onto two separate chloroamphenicol petri dishes
    4. For eppendorf 4, plate 200ul and 20ul onto two separate chloroamphenicol petri dishes
• Incubate the plates at 37'C overnight