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. |
