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  Section: General Biotechnology / Genes & Genetic Engineering
 
 
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Techniques of Genetic Engineering

 
     
 

Transfer of Recombinant DNA into Bacterial Cell
Once a mixture of recombinant DNA is obtained it is allowed to be taken up by the suitable bacterial cells. Originally the transformation procedure was developed by Mandell and Higa (1979). The strains of E.coli usually do not have restriction systems, hence it degrades foreign DNA. To escape from degradation exponentially growing cells are pretreated with CaCl2 at low temperature; thereafter DNA is mixed up. The event of entering the plasmid containing foreign DNA fragment into a bacterial cell is known as 'transformation'.

For the first time phage λ was used to transfer the foreign DNA into E. coli cell, therefore, it is often termed as transfection (a hybrid of transformation and infection). The efficiency of transformation is not high as it is influenced by bacterial strain and size of foreign DNA. It has been found that the efficiency of this process could generate about 105 transformants per mitrogram (Hg) of cloned circular plasmid (e.g. pBR322). It has not been possible to achieve efficiencies of over 108 transformants per g plasmid. If found that the efficiency of this process could generate about 105 transformants per microgram (mg) of cloned circular plasmid (e.g. pBR 322). It has not been possible to achieve efficiencies of over 108 transformants per g plasmid. If linear DNA is transformed it is almost completely insufficient in transformation.
 

Content

Gene cloning in prokaryotes

 

Isolation of DNA to be cloned

 

Insertion of DNA fragment into vector 

 

 

Use of restriction Linkers

 

 

Use of homopolymer tails

 

Transfer of recombinant DNA into bacterial cells

 

Selection of clones

 

 

Colony hybridization techniques

 

 

In vitro translation technique

 

 

Immunological tests

 

 

Blotting Techniques

 

Recovery of cells

 

Expression of cloned DNA

 

 

Shine-Dalgano sequence

 

 

Expression vectors

Gene cloning in eukaryotes

 

Plant cells

 

 

Yeasts

 

 

Filamentous fungi

 

 

Agrobacterium plasmids

 

 

Plant cell transformation

 

 

Plant cell transformation by ultrasonication

 

 

Liposome mediated gene transfer

 

Animal cell  

 

 

Animal viruses

 

 

Electroporation

 

 

Particle bombardment

 

 

Microinjection

 

 

Direct transformation

Site directed mutagenesis

 

Methods of mutagenesis

 
     
 
 
     



     
 
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