A Procedure for Making Chemically Competent Cells Using Calcium Chloride Solutions


In my experience, this has proven to be the most reliable method for making competent E. coli cells.  There are other methods that may be quicker, but accuracy can be fairly well assured when following this procedure.  I do not believe that this method has appeared in any textbooks in exactly this form; however, I have used it many time with nearly unwavering success.


In order to follow this method, you will need:


1) 0.1 M CaCl2


2) 0.1 M CaCl3 with 15% glycerol


3) LB media


All media and solutions should be autoclaved prior to beginning this procedure in order to avoid contamination of the cultures.


The procedure is as follows:


1) Place one colony of the appropriate cells in 10 mL LB media with any necessary antibiotic.


2) Allow cells to grow overnight at 37oC, preferably while shaking.


3) Add 5 mL of the overnight culture to 250 mL of LB media with any necessary antibiotic.


4) Allow the cells to grow at 37oC with shaking, until an OD600 of between 0.4 and 0.5 is reached. (this may take about 2 hours)


5) Transfer the cells to 50 mL plastic centrifuge tubes, and place the cells on ice for 20 minutes.


6) Centrifuge the cells at 4oC for 10 minutes at 2500 rotations per minute.


For the remainder of the procedure, make sure the cells are kept cold!  Be sure to always transport them on ice from now on.


7) Pour off the supernatant media, and resuspend the cells in 30 mL of 0.1 M CaCl2 that had been kept at a temperature of -20oC.


8) Keep the cells on ice for 30 minutes. 


9) Centrifuge the cells at 4oC for 10 minutes at 2500 rotations per minute.


10) Pour off the supernatant, and resuspend the cells, by pipetting, in 8 mL of 0.1M CaCl2 that contains 15% glycerol and has been kept at a temperature of -20oC.


11) Aliquot the suspension into 1.5 mL micro-centrifuge tubes in quantities of 200 microliters each.


12) Freeze the cells immediately in liquid nitrogen.


13) Store the cells at -80oC.


These cells can be used in transformations for about 6 months.


Good luck with your experiments!  I hope that having this tutorial available makes someone’s laboratory work go just a little bit more smoothly.  Having step by step instructions would have helped me quite a lot when I first started out.