Melbourne/Ligation Protocol

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  • Applications: Ligate DNA fragments together


  • Time to complete protocol: Overnight, 2 hours, or 10 minutes
    • Lab time: 5 minutes
    • Waiting time: Overnight, 2h, 10min
  • Approximate cost of materials: $

Contents

Method from primary and secondary reagents

Primary & secondary Reagents Required including controls
  • Ligase buffer (10X or 2X)
  • DNA for ligation
  • T4 DNA ligase
  • milliQ water
Method including controls

Add to a sterile 1.7ml Eppendorf

  1. Roughly an equal quantity of DNA fragments to be ligated together (usually 5ul of both vector and insert if gel-purified)
  2. 2ul of 10X buffer or 10ul of 2X buffer
  3. 1ul of T4 DNA ligase
  4. milliQ water to make up to 20ul

Incubate Overnight (if using 10X buffer) at 4 degrees, 2h or 5min (if using 2X buffer) at room temprature.

Equipement Required
  • 1.5mL Microfuge tubes
  • Pipettes
  • Fridge
References