Editing Electrotransformation of Vibro

== Electroporation protocol ==

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

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

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

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

<br/>4. Incubate the DNA and cell mixture on ice for 3–5 minutes.

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

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

<br/>6. Transfer cells to a chilled cuvette using a pipette. Verify that the cells completely occupy&nbsp;the bottom of the cuvette and that no air remains between walls of the cuvette and the&nbsp;cells.

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

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

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

<br/>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)&nbsp;at '''30°C for 2''' hours at to allow recovery of cells and expression of antibiotic resistance&nbsp;marker.

<br/>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&nbsp;temperature.&nbsp;