Transformation of cells using electroporation

Goal

To introduce plasmids into a bacterial strain.

Materials & Equipment:

Procedure:

If there are electrocompetent cells available to you in the -80°C, thaw the cells on ice and begin. If you need to make a fresh batch of electrocompetent cells, use either the small-scale or large-scale prep.

    1. Chill cuvettes for at least 5 minutes on ice.
    2. Add 375 uL of super pure, chilled water to each tube of electrocompetent C43(DE3) cells, to bring the final volume to 400 uL.
    3. Split the 400 uL into 4 tubes each containing ~100 uL.
    4. Set electroporator to 2.5kV. (Our electroporator is set to 600Ω)
    5. Add DNA to cells.
        For single transformations, add 5pg – 0.5ug DNA to cells.

          For double transformations, add 100 ng of each plasmid to cells.
    6. Mix by pipetting. Pipet into a sterile e.p. cuvette.
      • Note: This DNA should be salt-free, so when purifying with kit, use water.
      • Note: Include enough samples for +/- PCR.
      • Note: The DNA should not sit in the cells for more than 1 minute.
    7. Using a kimwipe, wipe the cuvette dry to prevent arcing!
    8. Place the DRY cuvette into the sample chamber. Apply the pulse by pushing the pulse button twice. Remove the cuvette.
      • Note: If you hear a popping sound, this means that the sample has arced. This could either be (1) too much salt in your sample or (2) a wet cuvette.
    9. Immediately add 1mL of room-temp SOC or LB and transfer to a culture tube
    10. Incubate 1 hr with shaking at 37°C.
    11. Plate out cells on LB+antibiotic. Grow o/n at 37°C.

For triple-plasmid transformations (Greg’s secret method):

    1. Chill 975uL SOC media in 1.5mL eppendorf tube and cuvettes on ice for at least 10 minutes.
    2. Thaw a tube of electrocompetent C43(DE3) cells. When cells are thawed (about 10 min), mix gently by flicking tube.
    3. Set electroporator to 1800V.
    4. Add 1uL of each plasmid to cells. Nucleic acid concentration should be about 100ng/uL.
    5. Flick tube gently to mix.
    6. Transfer cells to cuvette. Strike cuvette against bench top to get rid of bubbles and ensure cells are evenly spread along bottom of cuvette.
    7. Using a kimwipe, wipe the cuvette dry to prevent arcing!
    8. Take up the chilled SOC media with a pipette and hold it ready.
    9. With your other hand, place the DRY cuvette into the sample chamber. Apply the pulse by pushing the pulse button twice. Remove the cuvette as soon as you hear a beep.
    10. Immediately add the SOC media from the pipette and pipette up and down 2 times.
    11. Transfer the solution to the 1.5mL eppendorf tube SOC media was chilled in.
    12. Carry the cells over to incubator on ice.
    13. Tape the tube sideways at the bottom of the incubator and incubate the cells for 1 hr with shaking at 37°C and 275rpm.
    14. While cells are incubating, make plates by spreading 200uL of appropriate antibiotics for a final concentration of 1x and letting dry for at least 45min.
    15. Plate out cells on plates. Grow overnight at 37°C.

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