Competent E. coli cells:
This method produces competent E. coli cells that become susceptible to DNA transformation (Mandel and Higa, 1970; Oishi and Cosloy, 1972). A 5ml culture of XL1-blue bacterial cells in LB was grown to saturation over night at 37⁰C. This culture was then diluted 100-fold in LB and grown at 37⁰C to O.D. A600 of 0.25. Cells were chilled on ice for 5 min before being pelleted via centrifugation at 4,000 rpm for 5 min. The supernatant was discarded and the pellet resuspended in pre-chilled CaCl2. Cells were left on ice for a further 20 min before being pelleted again at 4,000 rpm for 5 min. Once the supernatant had been discarded, cells were finally resuspended in pre-chilled CaCl2 and left on ice for up to 24 hours (Dagert and Ehrlich, 1979). Glycerol was added to the sample to 16% concentration and competent cells were snap-frozen in liquid nitrogen in 100ul aliquots before being stored at -80⁰C until required.