E. coli Transformations using Electroporation
A negative control for your experiments should be transformed alongside your samples.
- Get required amount of electrocompetent cells from the -80°C freezer and let them thaw slowly on ice. Usually 1 tube (50μL / tube) per transformation.
- Take the appropriate amount of electroporation cuvettes from the -20°C freezer (1 per transformation) and label them accordingly
- Place 1-2μL of the DNA you wish to transform against the side of the cuvette, between the electrodes.
- Add your competent cells to each transformation by targeting the drop of DNA previously deposited.
- Keeping the cuvettes on ice, add 50μL of the competent cells and mix by pipetting up and down trying to avoid creating air bubbles
- Take your ice bucket, rich media (2YT or SOC etc.), a P1000 pipette and according tips to the Electroporator machine
- Set the electoporator to settings "Bacteria" and "ms"
- Thoroughly dry the cuvette external walls using paper to ensure a good connection
- Place the cuvette in the slideable holder with the cuvettes protuberance towards the electrodes (slide the extra bit of plastic in the separation between each half of the slider)
- Electroporate by pressing the lighting bolt button and record the "ms" value that appears. Ideally, the value should lie between 4 and 6.
- Quickly add 2-3mL of rich media to your cuvette, resuspend gently by pipetting up and down and let sit at room temperature until you are done with all the cuvettes
- Transfer the contents of the cuvette to a labeled eppendorf tube and incubate, shaking at 37°C for 30-45 minutes
- Plate the contents of the eppendorf onto appropriately selective media using a spreader and incubate at 37°C overnight
- The following day, check for colonies on the plates. You should often allow a minimum of 16 hours for the colonies to appear.