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Revision as of 00:53, 25 September 2012

Protocols  |  Single Gene Plasmids and Enzyme Characterisation  |  Showcasing Polysaccharide Production  |  The 3-Gene Inducible Plasmid Operon Construction  |  Glycobase

Linearized Plasmid Backbone Digestion and Ligation Protocol

For digestion of the linearized plasmid backbone, make an enzyme master-mix of: 5 µL NEB Buffer 2 0.5 µL BSA 0.5 µL EcoRI-HF 0.5 µL PstI 18.5 µL dH20 Then add 4µL of linearized plasmid backbone (25ng/µL for 100ng total) into 4µL of enzyme master mix. Digest for 37C/30 min and then heat kill at 80C/20 min. Once heat killed, a ligation mix is made-up containing: 2µl of digested plasmid backbone (25ng) Equimolar amount of EcoRI-HF SpeI digested fragment (< 3 µL) Equimolar amount of XbaI PstI digested fragment (< 3 µL) 1 µL T4 DNA ligase buffer. 0.5 µL T4 DNA ligase Top up solution to 10µL with water Ligate at 16C/30 min and then heat kill at 80C for 20 min. Transform with 1-2µL of product.