Question

1. Why are competent cells so fragile that they need to be handled carefully?

Answer 1

Preparation of competent cells In the Simple Cloning Lab we follow the simplest protocol for preparation of competent E. coli cells. The strain used is the strain DH5a. This method results in cells showing a transformation efficiency of 10 to 100 transformants per ug of plasmid DNA added. For routine cloning this is sufficient. The protocol starts with inoculation of liquid rich growth medium (e.g. LB) with 2% of a fresh overnight E.coli culture; so, for example, to 50 ml of LB 1 ml of the overnight culture is added. After growing at 37°C the absorbance (Optical Density at 600 nm.i.e. the light scattering measured in a spectrophotometer) is between 0.3 and 0.5 (the cells are now in the so-called mid-log phase). The cells are concentrated by centrifugation and resuspended very gently and in ice, in 1/20 volume of ice-cold 50 mM Calcium Chloride. They are left in ice for 2 to 24 hours to become competent, i.e. able to take up foreign DNA. The cell suspension maybe brought to 15 to 20% glycerol to survive a long term storage at -80°C. The calcium cations are thought to neutralize negatively charged phosphate in the cell membrane and the cells become very fragile. At this stage, DNA could now be added to these cells. The CaCl2 will precipitate the DNA, partly on the membrane, and by a heat shock treatment this foreign DNA would then be taken up by the cells.