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From 2013.igem.org

1. Ran the gel from the 26/07/2013 again for 45min
2. Digestion of plasmid backbone (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

1. Digestion of the limonene biobrick plasmid backbone (BBa_K118025)

• Using clone 1.1 purified plasmid DNA
• For the reaction adding:
  • 5ul NEB Buffer 3.1
  • 2ul of EcoRI
  • 2ul of PstI
  • 2.5ul of dH2O
  • 38.5ul of DNA
• digest at 37C for 30min
• heat kill at 80C for 20min

1. Ligation of insert to vector

• Calculate appropriate vector:insert ratio and convert to a 1:1 ratio
• Add the following substances
  • 11.1ul 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
• transform it into competent cells (E.coli)