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Clostridium botulinum

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.509 12/2001 Microorganism Clostridium botulinum Cell type Bacteria, gram positive Molecules injected Plasmid DNA (pGK12 in water) Growth medium TPGY medium Washing solution 10% PEG 8000 Electroporation solution 10% PEG 8000 Outgrowth medium TPGY medium with 25 mM MgCl2 Cuvette 4 mm gap width Reference Zhou Y. and Johnson E. A. 1993 Biotechnology Letters 15, No. 2 121-126 Making electrocompetent cells:

1. Grow cells in TPGY medium at 37 C to an O.D.660 of 0.8. 2. Harvest by centrifugation. Wash in 0.8 volumes of PEG at 4 C, keep cells cold during the entire procedure. 3. Resuspend cells in 0.8 ml 10% PEG (number of cells: 4 x 108 cells/ml), keep on ice.

Electroporation of cells:

  1. Add 1 g plasmid DNA to 800 l of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette. Keep on ice for 2 min.
  2. Wipe moisture from the cuvette and insert the cuvette into the device.
  3. Electroporation:

    Mode Prokaryotes O Voltage (V) 2,500 V Time constant (T) 5 ms
  4. Dilute cells into 10 ml TPGY medium with 25 mM MgCl2 and incubate 5 hours at 34 C.
  5. Plate cells on selective TPGY agar plates; incubate at 34 C for 2-3 days.
Expected Results: Transformation efficiency up to 3 x 103 transformants/g of DNA.


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