The most popular reagent for making E.coli competent cell is calcium chloride. The complete procedure of transformation is given in Figure 20.3 and it has following steps:
1. Bacterial Culture - The growth stage of the bacteria has a significant impact for its ability to take up foreign DNA. The bacterium at log phase is more active and efficient to perform DNA damage and repair than stationery phase. As a result, it is preferred to use bacteria of log phase for making competent cells for transformation.
2. Preparation of Competent Cell- Bacteria is incubated with divalent cation (Calcium chloride,Manganese chrloride or Rubidium chloride) for 30mins at 40C. During this process, cell wall of treated bacteria swells and it gather factors required for intake of DNA docked on the plasma membrane.
3. On the day of transformation, competent cells are incubated with DNA or circular plasmid containing appropriate resistance gene such as ampicillin resistance gene for 30mins on ice.
4. Heat Shock- Competent cells are given a brief heat shock (42°C for 90 sec) to relax the cell wall and high temperature stress causes upregulation of the factor responsible for DNA recombination and repair.
5. A chilled media is added for faster recovery of transformed cells.
6. it is plated on the solid media with appropriate antibiotics such as ampicillin and allowed to grow for another 18-24 hrs.
7. Transformed cells with appropriate resistance will grow and give colony.