1. Restriction Enzyme Vector Ligase Enzyme Recombinant DNA DNA Construct Digestion ligation

2. Electroporation in cloning presented by:vidahomayouni

3. DNA cloning The recombinant plasmids are then mixed with bacteria which have been treated to make them “competent”, or capable of taking in the plasmids This insertion is called transformation There are two methods for transforming E.coli cells with plasmid DNA; Chemical Transformation Electroporation There are two methods for transforming E.coli cells with plasmid DNA; Chemical Transformation Electroporation

4. Most efficient method of transforming bacteria A strong electrical impulse renders bacterial cell walls transiently permeable Efficiency: 10⁷ to 10¹⁰colonies per μg DNA Salts used in vector preparation may interfere with the electroporation process Washed E. coli are mixed with plasmid DNA. The E.coli + plasmid mix is then placed into a plastic cuvette. A short electric pulse is applied to the cells causing small holes in the plasma membrane through which the plasmid enters. Most efficient method of transforming bacteria A strong electrical impulse renders bacterial cell walls transiently permeable Efficiency: 10⁷ to 10¹⁰colonies per μg DNA Salts used in vector preparation may interfere with the electroporation process Washed E. coli are mixed with plasmid DNA. The E.coli + plasmid mix is then placed into a plastic cuvette. A short electric pulse is applied to the cells causing small holes in the plasma membrane through which the plasmid enters.