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From 2013.igem.org

Contents 1 Digestion of the chlorophenicol plasmid vector pSB1C3 2 Ligation of the plasmid pSB1C3 with the purified limonene biobrick (plasmid pSB1C3) 3 Transformation of the plasmid pSB1C3 and the limonene biobrick BBa_K118025 4 Gel electrophoresis

Digestion of the chlorophenicol plasmid vector pSB1C3

• Three different reactions
• Master Mix (changes were made from the protocol)
  • 5ul NEB Buffer 3.1
  • 0.5ul of EcoRI
  • 0.5ul of PstI
  • 19ul of dH2O
• For each reaction add:
  • 4ul of linearized backbone
  • 4ul of enzyme master mix
• digest at 37C for 30min
• heat kill at 80C for 20min

Ligation of the plasmid pSB1C3 with the purified limonene biobrick (plasmid pSB1C3)

• For this experiment, two separate samples will be run; a control and the actual ligation.
• Calculate appropriate vector:insert ratio and convert to a 1:1 ratio
  • volume of vector - 2ul
  • volume of insert - 0.8ul
• Add the following substances
  • 11.2ul of dH2O
  • 1ul of QS Ligase *
  • 5ul of 4xQS Buffer (vortex before use)
  • Mix thoroughly by pipetting
• Incubate at room temperature for 5 min to create cohesive ends
• Run 2.5-5ul of the ligation mixture onto an agarose gel to check ligation efficiency against a known marker. Add 1ul of dye to aid the visualization of results.
• Transform it into competent cells (E.coli)

*For the control, no QS Ligase is added but its equivalent volume (1ul) is made up by dH2O.

Transformation of the plasmid pSB1C3 and the limonene biobrick BBa_K118025

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 a LB (luria broth) petri dish
    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

Gel electrophoresis

• Gel electrophoresis was done to confirm the presence of the purified DNA.
• 5ul of DNA was run, but after electrophoresis no band was present.
• We assumed this was down to pippeting error and thus carried on with the transformation.

*Transformation has worked as shown on 02/08/2013