Competent cells Preparation Aim: To prepare competent cells from the given E.coli cells by calcium chloride method. Principle: Treatment of E.coli cells harvested at 0.6 OD with the ice cold solution of divalent cations (CaCl) induces a transient state of competence. The positively charged divalent cations interact with negatively charged DNA. The DNA uptake from the extracellular source is enhanced by sudden heat shock given to the chilled cells. Materials required: Eccoli strain, LB broth, CaCly solution, 15% glycerol, Micropipettes, micropipette tips, vortexer, refrigerator, centrifuge, incubator Procedure: 1. A loopful of E.coli strain was inoculated onto 100 ml of LB broth and incubated at 37°C with agitation to obtain 0.6 OD and the culture was chilled in ice for 30 minutes. Nv 1.5 ml of culture was aliquot into 2 sterile microfuge tubes using sterile micropipette tips. 3. The cells were pelleted by centrifugation at 4000 rpm for 10 minutes at 4°C. 4. The supernatant was removed by aspiration with pipette and was suspended with 300 jul of ice cold CaCl solution. The cells were incubated in ice for 15 minutes. 5. The cells were pelleted by centrifugation at 4000 rpm for 10 minutes at 4°C. 6. The supernatant was removed by aspiration and was suspended in 700 1] of ice cold CaCl. The cells were incubated in ice for 30 minutes.7. The cells were pelleted by centrifugation at 4000 rpm for 10 minutes at 4°C. 8. The supematant was removed by aspiration. For long term storage of competent cells was the pellet was resuspended in 100 jt] of CaClg + glycerol mix and store at -20°C or °C Observation: Competent cells were prepared and the cells were resuspended in CaCl and glycerol mix or further use. Result: Competent cells were prepared by using calcium chloride