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Brucella abortus

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.508 12/2001 Microorganism Brucella abortus Cell type Bacteria, gram negative Molecules injected Plasmid DNA (pBA.sodknr) Growth medium Trypticase soy broth Washing solution Sterile, cold water Electroporation solution 10% glycerol Outgrowth medium Trypticase soy broth Cuvette 2 mm gap width Reference Tatum F.M., et al 1992 Infection and Immunity 60 2863-2869

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.



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