Electroporation of freshly plated E. coli, P. aeruginosa, or Salmonella cells


Elisabeth Kugelberg

Cell Preparation
1. Streak a single colony on a LB-plate and keep at room temperature.
2. Fill a 1.5 mL eppendorf tube with 500 µL sterile water.
3. Add approximately 3 mg of cells, 3 generous swipes across the plate. It is important not to scrape the plate since it inhibits transformation.
4. Centrifuge at 14000 rpm 1 minute. Remove supernatant, suspend cells by pipet in 500 µL water.
5. Centrifuge again at 14000 rpm for 1 minute. Remove supernatant, suspend cells in 40 µL of water and keep on ice.
6. Cells are now ready for electroporation.

Electroporation
1. Add 5-50 nG of plasmid DNA and mix gently to ensure homogenous suspension.
2. Transfer DNA/cell suspensions to electroporation cuvettes and keep on ice.
3. After pulsing, immediately add ice-cold LB (or SOC media) to each cuvette.
4. Transfer to culture tubes and incubate for 1 hour at 37°C.
5. Spread on selective plates.

References
Enderle, P.J. & Farwell, M.A., "Electroporation of freshly plated E. coli and P. aeruginosa cells". Biotechniques. 1998 Dec; 25(6):954-956

Last Update: Thursday, 19-Jun-2014 11:54:31 PDT
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Eric Kofoid eckofoid at ucdavis.edu