西亚试剂：：Electroporation of P. aeruginosa

Electroporation of P. aeruginosa

1. Preparation of Electrocompetent Cells

    

   1. Inoculate a 5-ml of L-broth with cells of the P. aeruginosa strain to be electrotransformed. Grow culture overnight at 37°C on a roller.

       

   2. Transfer 2 ml of the overnight culture to 200 ml of fresh L-broth in a 1-liter sidearm flask. Grow this culture at 37°C with shaking (200 rpm) until OD540 = 0.3-0.5.

       

   3. Aliquot 35-40 ml of culture to each of four sterile Oak Ridge centrifuge tubes.

       

   4. Pellet the cells by centrifugation at 7000 x g (8000 rpm in SS-34 rotor) at 4°C for 10 minutes. Discard supernatant.

       

   5. Resuspend and wash the cells in an equal volume (35-40 ml) of ice-cold sterile 300 mM sucrose. Repellet the cells by centrifuging as before. Discard supernatant.

       

   6. Resuspend and wash the cells once again in 0.5 volume (18-20 ml) ice-cold 300 mM sucrose. Centrifuge as before. Discard supernatant.

       

   7. Resuspend the pellet in 0.01 volumes (350-400 µl) ice-cold 300 mM sucrose. The cell density at this point should be around 1 x 10(exp)11 cfu/ml.

       

   8. Chill the cells on ice for 30 minutes. P. aeruginosa cells thus prepared are ready for electroporation. Unlike E. coli, these cells cannot be electroporated with high efficency after being frozen.

       

    

2. Electroporation of the cells.

    

    

   1. Set the electroporation apparatus to 1.6-2.5 kV, 25 µF. Set the pulse controller to 200 omega.

       

   2. Add 1-5 µl plasmid DNA to tubes containing 40 µl of electrocompetent cells on ice. Mix by swirling with pipette tip. Transfer the DNA and cells to a pre-chilled electroporation cuvette (0.2 cm electrode gap) using a narrow pipette tip. Wipe any ice or water from sides of cuvette using a Kimwipe. Place the cuvette into the sample chamber.

       

   3. Energize the electroporation apparatus and deliver the pulse by pushing in both charging buttons simultaneously and holding until a short beep is heard. Note the time constant of the pulse and the actual voltage delivered.

       

   4. Remove the cuvette from the sample chamber. Add 3 ml L-broth and transfer the cells to a sterile polypropylene culture tube using a glass Pasture pipette. Incubate cultures for 2 hours at 37°C on a roller or with moderate shaking to allow for plasmid expression.

       

   5. Plate aliquots of the electroporation mixture on L-agar plates supplemented with the appropriate antibiotics. Incubate plates at 37°C.

       

   300 mM Sucrose