In these procedures, E. coli host cells are made competent and then transformed with either uncut pGEX DNA or recombinant pGEX DNA.
If electroporation is used to transform the cells, see Appendix 3 (Electroporation). Otherwise, proceed as described below.
Transform 1 ng of uncut (supercoiled) vector DNA in parallel with recombinant pGEX ligations to determine the efficiency of each competent cell preparation.
This protocol is based on the procedure of Chung et al. (Chung, C. T. et al. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. the USA 86, 2172–2175 ).1
All steps in this procedure should be carried out aseptically.
Use double-distilled water for the preparation of all solutions.
It is critical that the absorbance is not more than 0.5. This will take approximately 3 to 6 h.
Prewarming the broth to 37 °C will shorten the growth time.
Cells must be used for transformations within 2 to 3 h.