Team:INSA-Lyon/Protocols/Ligation

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Protocols

  1. Competent cells
  2. Transformation
  3. DNA extraction
  4. Digestion
  5. Ligation
  6. Measure of temperature and shaking speed influence
  7. Measure of osmotic pressure influence
  8. Granules extraction and intein cleavage
  9. Biofilms quantification
  10. Extra

    Ligation


      If it follows a digestion without purification: thermoinactivation 20 min at 70°C

    • Add x µL DNA (the entire digestion gel purification for part ligation) Plasmid: 50 to 200 ng; Insert: 100 ng (0,5 kb) to 1 µg (10 kb))
    • Add 2 µL Buffer T4 DNA ligase (Attention: this buffer contains ATP, defrost on ice)
    • Add 0,5 µL T4 DNA ligase (0,5 U) --> Add the enzyme last
    • Add sterile water qs 20 µL

      • Incubate 3h at room temperature or overnight at 15°C

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