Electroporation-PDF

This protocol is for transforming plasmid DNA into Escherichia coli cells.

Materials

• Electrocompetent cells
• Plasmid DNA (from a ligation reaction)
• Ice
• Ice bucket

For the following, you need one per DNA sample

• Electroporation cuvette (either 1mm or 2mm gap width)
• Electroporator
• 1.5 mL eppendorf tube
• LB-agar plate with appropriate antibiotic
• 1mL SOC at room temperature

Procedure

1. Chill electroporation cuvettes, DNA samples, and tubes on ice.
2. Place LB-agar plates in a 37°C incubator to warm.
3. Once cuvettes are cold, remove electrocompetent cells from the -80°C freezer and thaw on ice. Alternatively, freshly prepared electrocompetent cells may be used immediately.
4. If electrocompetent cells are not already in individual aliquots, then aliquot out into pre-chilled 0.6mL tubes.
5. Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes) or 2.5 kV (2mm cuvettes).
6. Dial a P2 pipeman to either 1 or 2μL depending on the salt content of your DNA sample. Use 2μL for samples that have been purified in some way.
7. Dial a P200 pipeman to 50μL or whatever volume of electrocompetent cells you want to use. Usually 20-50μL.
8. Dial a P1000 pipetman to 950μL and pipet in SOC. Place the pipeman on counter such that tip doesn’t touch anything.
9. Pipet 1-2μL of DNA sample and add to electrocompetent cells. Swirl the tip around gently in cells to mix DNA and cells. Do not pipet up and down.
10. Place cells back on ice to ensure they remain cold.
11. Transfer cell-DNA mixture to cuvettes using a P200 pipetman. Try not to handle the cuvette base too much so that it stays cold.
12. Tap the cuvette on the counter gently so that cells are at the bottom and remove any air bubbles.
13. Wipe off excess moisture from outside of the cuvette.
14. Place in the chamber of electroporator.
15. Slide the chamber in so that the cuvette sits snugly between electrodes.
16. Pulse the cells with a shock by pressing the button on the electroporator.
17. Remove the cuvette from the chamber and immediately add SOC. This step should be done as quickly as possible to prevent cells from dying off.
18. Transfer the SOC-cell mixture to a chilled Eppendorf tube.
19. Chill the sample on ice for 2 minutes to permit the cells to recover.
20. Transfer the Eppendorf tube to a 37°C incubator and shake to promote aeration. Incubate for 1 hr to permit expression of antibiotic resistance gene.
21. Plate transformation onto a prewarmed LB-agar plate supplemented with the appropriate antibiotic. I generally plate 200μL but the appropriate plating volume depends on the efficiency of the transformation.
22. Incubate the plate overnight at 37°C.
23. Leave the remaining SOC-cell mixture on the benchtop overnight.
24. If you don’t have any transformants, plate the rest of the transformation in the morning.

Notes

If you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampicillin takes a while to be pumped into cells at a high enough concentration to have an effect.