Competent cells

Overview
Digestions
pGT4ΔB
Electrophoresis
DNA clean-up
Ligation
Fragment isolation
Competent cells
Transformation
Recombinants
pGTλ3758ΔH
Miniprepping
Blotting
Probe Labeling
Hybridisation
Probe Detection
PCR

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 DH5α. This method results in cells showing a transformation efficiency of 105 to 106 transformants per g 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 37C 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 -80C.
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.