How to make Competent Cells

 

Materials Needed

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200 ml CaCl2 Solution:

                  60 mM CaCl2 (8.82g per liter)

                  15% glycerol (150ml per liter)

                  10 mM PIPES (3.02g per liter), pH 7.0 (must adjust pH with lots of NaOH                                     so PIPES will go into solution)

                  Filter sterilize or autoclave.

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400 ml culture of DH5α E. coli in early or mid log phase

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Eight 50ml sterile plastic tubes (or 30ml Nalgene tubes)

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Ice bucket with ice

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Pipettors and tips, pipet pumps and sterile pipets

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Refrigerated centrifuge with proper rotor

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Lots of COLD microcentrifuge tubes (keep in freezer).

 

Procedure

 

1.      Inoculate DH5α E. coli cells into 50ml LB broth.  Grow overnight at 37C with shaking (250 rpm).

 

2.      Inoculate 4ml of the culture into 400ml pre-warmed LB broth.  Grow at 37C, with shaking (250 rpm), to an OD590 of 0.375 (~2 hrs).  This procedure requires that cells be growing rapidly (early to mid log phase).

 

3.      Aliquot the culture into eight PRECHILLED 50ml sterile plastic tubes (50 ml per tube) and leave the tubes on ice 5-10 minutes. 

 

4.      Centrifuge cells for 7 min at 1600g (3500 rpm for JA-17 rotor) at 4C.

 

5.      Remove supernatant and gently resuspend each pellet in 10ml ICE COLD CaCl2 solution.

 

6.      Centrifuge 5 min at 1100g (2900 rpm for JA-17 rotor) at 4C.

 

7.      Remove supernatant and gently resuspend each pellet in 10ml ICE COLD CaCl2 solution. Keep resuspended cells on ice for 30 min.

 

8.      Centrifuge 5 min at 1100g (2900 rpm for JA-17 rotor) at 4C.

 

9.      Remove supernatant and gently resuspend each pellet completely in 2ml ICE COLD CaCl2 solution.  Immediately aliquot 225μl into microcentrifuge tube.  Immediately freeze at -70C.  It is very important to keep the cells cold, so keep aliquots on ice until ready to freeze.

Date Modified: 12/16/2010

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