(1) plasmid isolation and purification · in the alkaline lysis method, cells are lysed and dna...
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PLASMID ISOLATION AND PURIFICATION
BCH462- Practical
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¡ Definition.
¡ Extra hereditary genetic element.
¡ Replicon.
¡ Symbiotic relationship with the host
¡ Serve as vector.
¡ Classes of plasmid.
¡ Applications:
ØMolecular cloning,.
ØGene therapy,.
ØDrug production.
ØMaking a large amount of proteins.
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¡ Plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms.
¡ In the laboratory, plasmids may be introduced into a cell via transformation.
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Serve as vector
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At least one R.E recognition sites
EcoR1
Bam H1
Hind III
e.g: ampicillin resistance gene
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¡ Is an essential step for many molecular biology procedures.
¡ In general, plasmid purification involved three steps:
1. Growth of the bacterial culture.
2. Harvesting and lysis of bacteria.
3. Purification of plasmid DNA.
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¡ Depending upon nutritional status, bacteria exhibit different growth patterns which include:
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¡ Bacteria are recovered by centrifugation and lysed by any one of many methods, including:
è treatment with detergents, alkali, organic solvents, and heat.
¡ The choice among these methods depends on three factors:
Ø The size of plasmid.
Ø The bacterial strain.
Ø The technique used to subsequently purify the plasmid DNA.
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¡ Unlike the procedures for purification of genomic DNA ?
¡ There are three basic methods of plasmid preparation:
Ø Alkaline lysis method.
Ø Lysis in the presence of detergent.
Ø Application of affinity matrixes for plasmid or proteins.
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¡ To isolate pure plasmid DNA from E. coli using alkaline lysis method.
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¡ In the alkaline lysis method, cells are lysed and DNA denatured by SDS and alkaline pH.
¡ The SDS will lyse the bacterial cell membrane and denature the proteins.
¡ Alkaline pH will denature the genomic DNA and the proteins too.
¡ Neutralization of the solution .
¡ Precipitation of protein-SDS complexes.
¡ Subsequently both complexes, DNA and protein, are removed by centrifugation leaving native plasmid molecules in the supernatant.
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¡ Concentration of plasmid DNA (ng/µl ) = _________________
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v What is plasmid copy umber ? How does it affect the isolation protocol?
v What is the importance of antibiotic gene in the plasmid?
v How plasmid could contribute to drug resistance in bacteria ?