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Streptococcus Salivarius

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Streptococcus salivarius
Multiporator / Electroporator 2510
Transformation Protocol Protocol No. 4308 915.541 – 03/2002
Microorganism Streptococcus salivarius
Cell type Bacteria, gram positive
Molecules injected Plasmid DNA
Growth medium HJGS (Hogg-Jago glucose broth with 0.4 sorbitol)
Washing solution 5 mM potassium phosphate (pH 4.5), 0.4 M sorbitol, 10% glycerol
Electroporation solution 5 mM potassium phosphate (pH 4.5), 0.4 M sorbitol, 10% glycerol
Outgrowth medium HJGS (Hogg-Jago glucose broth with 0.4 sorbitol)
Cuvette 1 mm gap width
Reference Buckley, N. D. et al • 1999 • Applied and Environmental Microbiology 65, No. 9 • 3800-3804
Making electrocompetent cells:

1. Cultivate cells by adding 1% overnight preculture to fresh medium. Grow cells at 37 °C without agitation to a cell
density of O.D.660 of 0.5. Add glycine to a final concentration of 10% and incubate again for 1 h at 37 °C.
2. Harvest by centrifugation.
3. Wash twice in ice-cold washing solution.
4. Resuspend in electroporation solution to a 50-fold concentration and freeze in an ethanol-dry ice bath. Store at -80 °C.

Electroporation of cells:

  1. Thaw cells on ice. Add 1 µg plasmid DNA to 100 µl of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette.
  2. Wipe moisture from the cuvette and insert the cuvette into the device.
  3. Electroporation:

    Mode Prokaryotes “O“
    Voltage (V) 1,600 V
    Time constant (T) 5 ms

  4. Add 1 ml ice-cold outgrowth medium and incubate for 3 h at 37 °C.
  5. Plate aliquots onto selective agar plates; incubate 2-3 days at 37 °C.
Expected Results:
Transformation efficiency up to 1 x 105 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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