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

 
     
 

Electroporation
Electroporation (using electric field) is now used to transfer the foreign DNA into the fragile cells. Brief pulses of high voltage electricity (about 350 V) is applied to protoplasts suspension containing naked or recombinant plasmids. The electric pulses induce the formation of large pores in the cell membrane. These pores give a passage through which the foreign DNA can enter into the protolasts and thus, increase the transformation frequency. The transformed protoplasts are cultured for about one month, These develop microcalli which are plated on solid medium containing selective marker (e.g. kanamycin). After 37-45 days, the calli are analyzed for the presence of differences in transformed cells.

By using electroporation method, genes of choice have been successfully transferred in protoplasts of wheat, rice, petunia, sorghum, maize and tobacco. Moreover, transformation frequency can be improved by using linear DNA instead of circular one, by giving heat sock to protoplast at 45°C for 5 minutes, by adding PEG and using 1.25 kV/cm voltage.
 

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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