From 2007.igem.org

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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 1 Method from primary and secondary reagents 1.1 Primary & secondary Reagents Required including controls 1.2 Method including controls 1.3 Equipement Required 1.4 References

Method from primary and secondary reagents

Primary & secondary Reagents Required including controls

• T4 Ligase buffer 10X or T4 Ligase buffer 2X
• DNA for ligation
• T4 Ligase
• milliQ water

Method including controls

Add to a sterile 1.7ml Eppendorf

• Roughly an equal quantity of DNA fragments to be ligated together (usually 5ul of both vector and insert if gel-purified)
• 2ul of T4 Ligase buffer 10X or 10ul of T4 Ligase buffer 2X
• 1ul of T4 Ligase
• 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

• microcentrifuge: 1.7ml
• Pipettes
• Fridge

References