restriction enzyme vector ligase enzyme recombinant dna dna construct digestion ligation

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.