Please explain Why do you add the CacL2 transformation solut

Why do you add the CacL_2 transformation solution? Step 6 in the protocol - why do you incubate the tubes on ice for 10 minutes? What would you expect to grow on the following LB nutrient agar plates? +pGLO on LB: -pGLO on LB/amp/ara: Why do you heat shock the cells?

Solution

1. Positively charged calcium ions of CaCl2 attract both the negatively charged DNA backbone and the negatively charged groups in the LPS inner core. The plasmid DNA can then pass into the cell .

2. To allow the Plasmid to stick on the surface of the cell so that after heat shock DNA can enter the cells.

3.

A) +pGLO add will give the transformed colony and non transformed cells here they did not use antibiotic resistance.

B) -pGLO addition will lead to no colony.

4.

The plasmid DNA can then pass into the cell upon heat shock, where chilled cells (+4 degrees Celsius) are heated to a higher temperature (+42 degrees Celsius) for a short time. That will disturb the membrane and create pores that facilitate the entry of DNA into the cell.