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