Help:Protocols/Ligation


Ligating a Part from iGEM Videos.


After following our restriction digest protocol (which uses 250ng of DNA) you may follow these steps for ligation.

Ligation

  • Add 2ul of digested plasmid backbone (25 ng)
  • Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 ul)
  • Add equimolar amount of XbaI PstI digested fragment (< 3 ul)
  • Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase
  • Add 0.5 ul T4 DNA ligase
  • Add water to 10 ul
  • Ligate 16C/30 min, heat kill 80C/20 min
  • Transform with 1-2 ul of product

Note: For linearized plasmid backbones provided by iGEM HQ, a plasmid backbone with an insert of BBa_J04450 was used as template. As a result any red colonies that appear during your ligation may be due to the template as a background. Digesting with Dpn1 before use should reduce this occurrence.