Team:BIOTEC Dresden/Protocols:Ligation



Try different molar ratios of vector to plasmid to increase ligation effficiency

The sticky ends of both the digested insert and vector DNA must be comptabile


  • ultra-pure water
  • purified digested insert DNA
  • purified digested vector DNA
  • 10X T4 DNA ligase buffer
  • T4 DNA ligase


  • How to calculate the Insert Amount, if, for example, a 6:1 molar ratio is decided upon
  • Into a 1.5mL add the following:
  • 2.0µL 10X T4 DNA Ligase Buffer
  • volume of insert that corresponds to the determined molar ratio
  • volume of vector that corresponds to the determined molar ratio
  • 0.5µL T4 DNA Ligase
  • Adjust final volume to 20µL with ultra pure water
  • Vortex and pulse spin down
  • Incubate at room temperature for 1 hour
Share to Twitter Share to Facebook Share to Orkut Stumble It Email This More...