For the transformation of pUC plasmids, DH5 
cells[114], an
E. coli strain, were used. Non-competent cells
were plated on an
agarose plate and incubated overnight. The next day, 50 ml
LB medium were inoculated with one colony.
The cells were grown with vigorous shaking at 37psy176 C to a density of
cells/ml. This usually takes 2-3 hours (OD 
0.3).
The cell suspension is centrifugated at 3000 g for 5 minutes at 4psy176 C.
The pellet is resuspended in 20 ml of 0.1 M CaCl 
and left on ice for 30
minutes. Then the suspension is centrifuged again at 3000 g for 5 minutes at
4psy176 C. The cells are now resuspended in 2 ml CaCl and the competent
cells stored for 2-3 days at 4psy176 C.
For maximum transformation efficiency, it is very important that the bacterial culture is in the logarithmic phase of growth and that cell density is low at the time of treatment with calcium chloride; and that the cells are maintained at 4psy176 C for 12-24 hours. During this period, the efficiency of transformation increases fourfold to sixfold[115].