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Lactococcus lactis

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.519 12/2001 Microorganism Lactococcus lactis MG1363 Cell type Bacteria, gram positive Molecules injected Plasmid DNA (pGK12) Growth medium Complex medium with 1% glycine Washing solution 0.5 M sucrose, 10% glycerol Electroporation solution 0.5 M sucrose, 10% glycerol Outgrowth medium Ice-cold complex medium with 0.5 M sucrose, 20 mM MgCl2, 2 mM CaCl2 Cuvette 1 mm gap width Reference Dr. Horst Neve Bundesanstalt fr Milchforschung Institut fr Mikrobiologie Hermann-Weigmann Str. 1 D-24103 Kiel Phone +49 431 6091 Fax +49 431 609222 Making electrocompetent cells:

1. Grow cells overnight at 30 C to an O.D.620 of 0.7. 2. Wash twice with ice-cold washing solution. 3. Resuspend cells in 1/100 volume of electroporation solution. Keep on ice.

Electroporation of cells:

  1. Add 0.25 g plasmid DNA (in water) to 100 l of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into 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. Add 1 ml ice-cold complex medium, incubate 2 hours at 30 C.
  5. Plate diluted cells on selective chloramphenicol plates. Incubate 2 days at 30 C.
Expected Results: Transformation efficiency up to 1.4 x 106 transformants/g of DNA.



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