Team:TU-Delft/Protocol 6



Our project deals with E.coli cells which sense Auto-inducing peptides (AIPs) from the Staphylococcus aureus and starts producing Antimicrobial peptides in order to kill the Staphylococcus aureus. Different protocols used during the project are described below.



  1. digested plasmid DNA or PCR product
  2. T4 ligation buffer (10x) (Fermentas)
  3. T4 ligase (Fermentas)
  4. H2O
  5. water bath at 16 °C


  1. Ligations (pasting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier.
  2. Reaction for one sample:
  3. Component Sample
    DNA insert x μL
    DNA vector x μL
    T4 Ligation buffer (10×) x μL (for 1×)
    T4 Ligase 1.0 μL
    H2O x μL
    10-15 μL

    The final concentration is preferably ~100 ng/μL. Incubate at 16 °C for at least 3 hours. For transformation use circa half of the ligation mix. Transform circa half of the ligation mix. Incubate at 16 °C o/n.