Preparation of E.Coli Competent Cells

Preparation of electro-competent cells of E. coli DH5-α

1.      A single colony from a freshly grown plate of DH5-α (GIBCO-BRL, USA) was picked and transferred into 100 ml LB medium in 1 liter flask and incubated at 37 ⁰C overnight with vigorous shaking.

2.      2.5 ml of the overnight culture was taken and transferred to 250 ml LB in 1000 ml flask and shaken vigorously at 37 ⁰C until O. D⁶⁰⁰ of 0.5-1.0 (10¹⁰ cells/ml).

3.      The cells were transferred aseptically to sterile disposable 50 ml propylene tube.

4.      The culture was cooled by keeping on ice for 10 minutes.

5.      The cells were pelleted by centrifugation at 5000 rpm at 4 ⁰C for 15 minutes and resuspended in one of volume sterile cold distilled water.

6.      The cells were pelleted by centrifugation at 5000 rpm at 4 ⁰C for 15 minutes and resuspended in 0.5 volume of sterile cold distilled water.

7.      The cells were pelleted by centrifugation at 5000 rpm at 4 ⁰C for 15 minutes and resuspended in 0.02 volume of sterile cold distilled water.

8.      The cells were again pelleted by centrifugation at 5000 rpm for 15 minutes and suspended finally in 0.002-0.003 volume sterile 10% cold glycerol.

9.      The cells were stored in aliquots of 50 μl or 100 μl at -70 ⁰C.