Team:INSA-Lyon/Protocols/Ligation

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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