( ...)Staphylococcus epidermidis

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.534 01/2002 Microorganism Staphylococcus epidermidis Cell type Bacteria, gram positive Molecules injected Plasmid DNA (pC194) Growth medium Basic medium (BM: 1% peptone, 0.5% yeast extract, 0.1% glucose, 0.5% NaCl, 0.1% K₂HPO₄) Washing solution 10% glycerol (in distilled water) Electroporation solution 10% glycerol (in distilled water) Outgrowth medium SMMP50 medium (5.5 parts SMM buffer (1 M sucrose, 0.04 M maleic acid,
0.04 M MgCl₂, pH 6.5), 4 parts 7% Panassy broth, 0.5 parts 10% BSA) Cuvette 2 mm gap width Reference Augustin J. and Gtz F. 1990 FEMS Microbiology Letters 66 203-208 Making electrocompetent cells:

1. Inoculate a flask of BM with a fresh overnight culture. Grow at 37 C with shaking to an O.D.₅₇₈ of about 0.5 to 0.65. 2. Wash in one volume of 10% glycerol at room temperature, followed by washes with 1/2, 1/20 and 1/50 volumes. 3. Resuspend to final volume of 1-5 x 10¹⁰ cells/ml in 10% glycerol. Aliquote and freeze at 70 C.

Electroporation of cells:

1. Thaw cells for 5 min at room temperature. Add 1-2 l plasmid DNA (100-500 ng) to 50 l of electrocompetent cells. Homogenize by gently mixing with pipette several times. Incubate at room temperature for 30 minutes.
2. Transfer mixture into a cuvette and insert the cuvette into the device.
3. Electroporation:
   
   Mode Prokaryotes O Voltage (V) 1,800 V Time constant (T) 5 ms
   
4. Add 1 ml SMMP 50 medium to the cells and transfer to sterile tube. Incubate 90 minutes at 37 C with moderate
   shaking.
5. Plate on selective BM plates. Incubate 20 hours at 37 C.

Expected results: Transformation efficiency up to 1.5 x 10⁵ transformants/g of DNA.


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