Material and Equipments:
1. E.Coli Competent Cells
2. Water Bath
3. Luria Bertani Media
4. LB-Agar Plate
5. Incubator
Procedure : The outline of the procedure involved in calcium chloride mediated transformation is given in the Figure 38.3.
1. 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.
2. Heat Shock-Competent cells are given a brief heat shock (420C for 90 sec) to relax the cell wall and high temperature stress causes upregulation of the factor responsible for DNA recombination and repair.
3. A chilled bacterial media is added for faster recovery of transformed cells.
4. it is plated on the solid media with appropriate antibiotics such as ampicillin and allowed to grow for another 18-24 hrs.
5. Transformed cells with appropriate resistance will grow and give colony (Figure 38.4).
Observation: The transformed LB agar plate in Figure 38.4 has 1488 colonies.
Calculation of the transformation efficiency:
If 10ng of plasmid gives ~1488 colonies.
Then 1µg of plasmid transform 1488 x102=1.488x 105 colonies
Therefore the transformation efficiency was found to be -1.4 x 106 colonies.
Figure 38.3: Steps in bacterial transformation by CaCl2 method.