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Pediococcus spp.

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.540 07/2002 Microorganism Pediococcus spp. Cell type Bacteria, gram positive Molecules injected Plasmid DNA (pGK12 and pPN-1) Growth medium MRS-G with 0.5 M sorbitol, 3 % glycine and 40 mM DL-threonine Washing solution 0.5 M sorbitol, 10 % glycerol Electroporation solution 0.5 M sorbitol, 1 mM K2HPO4, 1mM MgCl2, pH 7.0 Outgrowth medium MRS with 0.5 M sorbitol, 20 mM MgCl2 and 2 mM CaCl2 Cuvette 1 mm gap width Reference Caldwell, S. L. et al 1996 Applied and Environmental Microbiology 62, No. 3 936-941 Making electrocompetent cells:

1. Cultivate cells by adding 12 ml overnight preculture (grown in MRS-G with 0.5 M sorbitol) to 800 ml growth medium. Incubate for 2 to 4 hours at 37 C to a cell density of O.D.600 of 0.4-0.6. 2. Harvest by centrifugation. 3. Wash twice in 25 ml washing solution. 4. Resusp end in 1 ml electroporation solution.

Electroporation of cells:

  1. Add 400-600 ng plasmid DNA to 80 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) 1,800 V Time constant (T) 5 ms
  4. Immediately add 2 ml outgrowth medium and keep on ice for approx. 5 min. Incubate for 2 h at 37 C.
  5. Plate aliquots onto selective agar plates; incubate 2-5 days at 37 C.
Expected Results: Transformation efficiency up to 4.6 x 103 transformants/g of DNA.



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