Step 3: Transformation: transfer of recombinant plasmid DNA to a suitable host:

The ligation reaction mixture of recombinant DNA described in the step 2 is introduced into bacterial cells in a process called transformation. The traditional method to prepare cells for transformation process is to incubate the cells in a concentrated calcium salt solution to neutralize the negative charge of membrane (due to salicylic acid), so that the negatively charged DNA molecules can come close to bacterial membrane and during heat shock (method of transformation) can easily enter in the cells. These "competent" cells are then mixed with ligation product to allow entry of the DNA into the bacterial cell. An alternative mode of transformation is electroporation method which is used to drive DNA (comparatively larger size) into cells by a strong electric current. This method is not very common due to less percentage of survival of transformed cells.

As mentioned earlier bacterial species use restriction enzymes to degrade foreign DNA lacking the methylation pattern, including the plasmids, then why don't they degrade the transformed recombinant DNA. The answer is that molecular biologist have cleverly engineered and developed the bacterial strains that lake restriction-modification system. The best example is common lab strain E.coli DH5a. A transformed bacterial cell may carry either recombinant or nonrecombinant plasmid DNA. The plasmid DNA multiplies within each transformed bacterial cell. Each transformed bacterial cell when plated to the solid agar media (nutrient media) can multiply to form a visible colony made of millions of identical cells. As the transformed cell divides, the plasmids are passed on to progeny, where they continue to replicate. Single transformed bacteria undergo numerous cell divisions results in clones of a cell (single bacterial colony) from a single parental cell. From this step the name "cloning" is given. From the colony of bacterial cells the cloned DNA can now be isolated.

Step 4: Screening for transformed cells:

To avoid the growth of the untransformed bacterial cells, plasmid vectors are engineered with selectable marker gene for resistance to the antibiotics. The media in which the transformed bacterial cells are grown is supplied with that antibiotic whose resistance gene is present in the plasmid. Due to this only transformed cells show antibiotic resistance will grow in the media supplied with antibiotic and untransformed cells cannot grow as they do not carry antibiotic resistance gene. Transformed bacterial cells may contain either recombinant plasmid DNA (vector containing foreign DNA insert) or non-recombinant plasmid DNA (self ligated vector only). Both type of transformed bacterial cells will show antibiotic resistance and grow on the agar media plate.