Electroporation of Pseudomonas putida or Pseudomonas fluorescens

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1. Keep everything on ice throughout the entire procedure. Prechill cuvettes on ice.
   
   
2. Transfer 6mL of an overnight culture into a Falcon tube. Centrifuge at 2000 rpm for 10 minutes and remove the supernatant.
   
   
3. Resuspend the pellet in 4mL sterile, 1mM HEPES solution (containig 10% glycerol by volume) and Centrifuge again for 5 minutes at 2000 rpm.
   
   
4. Discard the supernatant, resuspend the pellet in 4mL HEPES (with 10% glycerol) and repeat the centrifugation.
   
   
5. Remove the supernatant and resuspend the pellet in 400 uL HEPES (10 % glycerol). Store this on ice until use.
   
   
6. Add the DNA and transfer to an ice-cold cuvette.
   
   
7. Electroporate with a BIO-RAD electroporator. Make sure the time constant is equal to or greater than 4.
   
   
8. Immidiately mix the cells with 1mL SOC and transfer to a prechilled Eppendorf tube. Shake at 20°C for 2 hours.
   
   
9. Plate #1: Plate 100 uL on selective medium at a 1:10 dilution.
   
   
10. Plate #2: Spin the remainder at full speed for one minute, remove the supernatant and resuspend the pellet in 100mL of SOC and plate on a selective medium.