Team:Cambridge/Protocols/DigestionLigation

This protocol is adopted from the Parts Registry: http://partsregistry.org/Help:Protocols/Linearized_Plasmid_Backbones. It is used to create biobricks which comply to BBF RFC 10.

Digest

Enzyme Master Mix for Plasmid Backbone (25ul total, for 6 rxns)

• 5 ul NEB Buffer 2
• 0.5 ul BSA
• 0.5 ul EcoRI-HF
• 0.5 ul PstI
• 0.5 ul DpnI (Used to digest any template DNA from production)
• 18 ul dH20

Digest Plasmid Backbone

Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)

Add 4 ul of Enzyme Master Mix

Digest 37C/30 min, heat kill 80C/20 min

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.