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Bifidobacterium Animalis

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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 µg plasmid DNA to 80 µl 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 µl 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/µg of DNA.

Contact Information

In the United States:
Eppendorf North America, Inc.
102 Motor Parkway,
Hauppauge, NY 11788-5178
Tel: 800-645-3050
Fax: 516-334-7506
Web Site: http://www.eppendorfna.com/

Outside the United States:
Eppendorf AG
Barkhausenweg 1
22339 Hamburg
Germany

Customer Service: ++ 49 40 53 801-0

Fax Number: ++ 49 40 53 801-556

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