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

• 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 equal quantity of DNA fragments to be ligated together (usually 5ul of both vector and insert if gel-purified)
2. 2ul of 10X buffer Italic textorItalic text 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