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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
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Restriction Enzyme Vector
Ligase Enzyme
Recombinant DNA
DNA Construct
Digestion
ligation
Electroporation in cloning
presented by:vidahomayouni
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
Electroporation
• 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.