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Bifidobacterium animalis

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.538 04/2002 Microorganism Bifidobacterium animalis Cell type Bacteria, gram positive Molecules injected Plasmid DNA Growth medium MRS broth with 0.05% cysteine.HCl and 0.5 M sucrose (final concentrations) Washing solution 0.5 M sucrose Electroporation solution 0.5 M sucrose, 1 mM ammonium citrate, pH 6 Outgrowth medium MRS broth with 0.05% cysteine.HCl and 0.5 M sucrose (final concentrations) Cuvette 1 mm gap width Reference Argnani, A. et al 1996 Microbiology 142 109-114 Making electrocompetent cells:

1. Cultivate cells by using an overnight culture to inoculate fresh medium. Grow cells overnight at 37 C. Dilute this culture 1:25 in fresh medium and cultivate at 37 C until an O.D.695 of 0.2. Chill bacteria on ice. 2. Harvest by centrifugation. 3. Wash twice with 0.5 M sucrose 4. Resuspend in about 1/250 of the original culture volume of 1 mM ice-cold sucrose-citrate buffer, dispense in tubes and incubate for 3.5 hours at 4 C.

Electroporation of cells:

  1. Add 0.5-1.5 g plasmid DNA to 80 l of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into prechilled cuvette.
  2. Wipe moisture from the cuvette and insert the cuvette into the device.
  3. Electroporation:

    Mode Prokaryotes O Voltage (V) 1,200 V Time constant (T) 5 ms
  4. Dilute with 800 l outgrowth medium and incubate for 2.5 h at 37 C.
  5. Plate onto selective MRS agar plates; incubate anaerobically for 2-3 days at 37 C.
Expected Results: Transformation efficiency up to 9.4 x 104 transformants/g of DNA.



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