Help:Protocols/Ligation

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Ligation

After growing up a part from a distribution or request, you'll want to miniprep it. At iGEM HQ, we use the following protocol.

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 [http://www.neb.com/nebecomm/products/productm0202.asp T4 DNA ligase buffer]. Note: Do not use quick ligase
• Add 0.5 ul [http://www.neb.com/nebecomm/products/productm0202.asp 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.