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Propionibacterium Freudenreichii

Propionibacterium freudenreichii
Multiporator / Electroporator 2510
Transformation Protocol Protocol No. 4308 915.527 – 04/2002
Microorganism Propionibacterium freudenreichii
Cell type Bacteria, gram positive
Molecules injected Plasmid DNA
Growth medium SLB medium
Washing solution Ice-cold 0.5 M sucrose
Electroporation solution Ice-cold 0.5 M sucrose buffered with 1 mM potassium acetate (pH 5.5)
Outgrowth medium SLB medium with 0.5 M sucrose
Cuvette 1 mm gap width
Reference Jore, J. P M. et al • 2001 • Applied and Environmental Microbiology 67, No. 2 • 499-503
Making electrocompetent cells:

1. Cultivate cells anaerobically at 30 °C in SLB medium to the stationary growth phase. Dilute 1:50 in fresh SLB medium and incubate again for about 20 h.
2. Harvest in the exponential growth phase by centrifugation and wash extensively with ice-cold 0.5 M sucrose.
3. Wash once in ice-cold electroporation solution.
4. Resuspend in ice-cold electroporation solution (about 1/100 of the original culture volume).

Electroporation of cells:

  1. Add plasmid DNA to 80-100 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) 2,000 V
    Time constant (T) 5 ms

  4. Immediately add 900 µl cold outgrowth medium and incubate for 2.5-3 h at 30 °C.
  5. Plate cells onto selective SLB plates; incubate 5-7 days at 30 °C under anaerobic conditions.
Expected Results:
Transformation efficiency up to 1 x 108 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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