Electrotransformation of Vibro

Electroporation protocol

1. For each transformation reaction,
• Thaw one vial of Vmax™ Express Electrocompetent cells on ice for 5 minutes.
• Warm one vial of Vmax™ Recovery Media to room temperature (20°C to 24°C).
• Pre-chill a 0.1-cm electroporation cuvette on ice.

2. Transfer 500 µL Vmax™ Recovery Media to a Falcon® 14-mL Round-Bottom Tube.

Allow media to sit at room temperature until needed in Step 8.
Note: Vmax™ recovery medium is rich and prone to contamination. Discard any remaining medium or return it to −20°C for storage.

3. As soon as the electrocompetent cells are thawed, add 500 pg – 5 ng of plasmid DNA to the competent cells on ice. Briefly mix by flicking tubes.

4. Incubate the DNA and cell mixture on ice for 3–5 minutes.

5. During incubation, verify the Gene Pulser® Xcell System is set to the following electroporation parameters: 700 V, 25 uF, 200 Ohms, 0.1-cm cuvette.

Note: These are the optimized electroporation settings for the Gene Pulser® Xcell™ Microbial System using a cuvette with a 0.1-cm gap. If other instruments and/or cuvettes are used, you may need to optimize the conditions for best results.

6. Transfer cells to a chilled cuvette using a pipette. Verify that the cells completely occupy the bottom of the cuvette and that no air remains between walls of the cuvette and the cells.

7. Transfer the cuvette to the shock pod and perform electroporation.
Note: Time constants typically range from 3.3–3.7 ms.

8. Transfer cells to a room temperature Falcon® 14-mL Round-Bottom Tube containing recovery media immediately after pulse.

9. To transfer any remaining cells, add 200 μL of Vmax™ Recovery Media to the cuvette.

Dispense the media to the same round-bottom tube from Step 8.

10. Place the Falcon® 14-mL Round-Bottom Tubes in a tube rack in an orbital shaker (250 rpm) at 30°C for 2 hours at to allow recovery of cells and expression of antibiotic resistance marker.

Note: Recovery can be performed at 37°C unless kanamycin is used for selection.

11. During cell recovery, pre-warm LB-Miller agar plates prepared with antibiotics at the appropriate incubation temperature.