Brucella Abortus

Brucella abortus

Making electrocompetent cells:

1. Grow cells at 30 °C to a density of O.D.₆₀₀: 1.5-2.0.


2. Harvest by centrifugation and wash five to seven times in sterile, cold water.


3. Resuspend the cells in 10% glycerol to a final concentration of 4-6 x 10¹¹ cell/ml and store frozen at –70 °C.


4. For electroporation frozen cells were thawed in ice and diluted with sterile water to a density of 1 x 10¹⁰ cells/ml prior to use.

Electroporation of cells:

1. Grow cells in Trypticase soy broth at 37 °C with vigorous shaking, chill on ice for 10 min.
   
2. Harvest by centrifugation (10 min, 10,000 x g). Wash three times in an equal volume of sterile cold water.
3. Resuspend cells in 1/500 volume of 10% glycerol (number of cells: 10¹⁰ cells/ml). Freeze aliquots on dry ice and
   store at –70 °C.
   
   

   Mode	Prokaryotes “O“
   Voltage (V)	2,500 V
   Time constant (T)	5 ms

   
   
4. Immediately add 1 ml trypticase soy broth, incubate 6 hours at 37 °C.
5. Plate cells on selective tryptose agar plates, incubate at 37 °C for four days.