Apr 03, 2025

Public workspaceMaking electrocompetent E. coli for plasmid transformation

This protocol is a draft, published without a DOI.
  • 1University of Warwick, School of Life Sciences
Rebecca Quinn
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Protocol Citation: Rebecca Quinn 2025. Making electrocompetent E. coli for plasmid transformation. protocols.io https://protocols.io/view/making-electrocompetent-e-coli-for-plasmid-transfo-d7bg9ijw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 02, 2025
Last Modified: April 03, 2025
Protocol Integer ID: 126024
Disclaimer
This is useful for common lab strains like E coli K12 MG1655 and B REL606
Abstract
"Quick and Dirty" way to make E coli strains electrocompetent on a lab bench.
Preparation
Preparation
13h 35m
13h 35m
Make an DurationOvernight culture of the cells you wish to make competent (24 hours etc)

12h 30m
Put the following in the freezer:
a) bottle of sterlie diH2O (don't let it freeze)
b) however many 2mm electro-cuvettes you need
1h
Get two or three boxes of ice
4m
Get some SOC media.
1m
Grow cells up
Grow cells up
1h 7m
1h 7m
Re-sub Amount100 µL into Amount5 mL LB in a universal

5m
Grow at Temperature37 °C on shaker forDuration01:00:00 -Duration01:15:00 (till about 0.3-0.4 OD600)

1h
Once at the right OD, put the cells on ice.
1m
Put the sterile diH2O onto ice.
1m
Preparing the cells
Preparing the cells
35m
35m
Spin down the Amount5 mL cells into an eppendorf tube, using a benchtop centrifuge at Centrifigation14000 x g . Tip the supernatant into the waste.
Keep the cell pellet on ice in between spins.

10m
Once all cells are pelleted, add Amount1000 µL of ice cold diH2O to the tube and resuspend the pellet. Don't worry if the pellet is on the smaller side.

1m
Spin down cells using a benchtop centrifuge at Centrifigation14000 x g .
1m
Using a pipette, remove the supernatant smoothly and carefully. Keep an eye on the pellet to make sure that it is not being sucked up in the pipette tip.
1m
Keep adding Amount1000 µL of ice cold diH2O to the tube and resuspending the pellet, spinning down, and removing the supertant with a pipette until the pellet appears "unstable". The more you go through this, your pellet should get paler. Wash, rinse, repeat.

Unstable pellet: the pipette tip will start pulling the pipette up with the suction. The best way to determine this is to remove the supernatant by putting the pipette tip close to the pellet and keeping a very careful eye on the cells to make sure they don't get shlurped up.
20m
When the pellet becomes "unstable", add Amount100 µL of ice cold diH2O to the tube. Resuspend the cells. Should be milky white. Keep on ice.

Note: if you think you've achieved this with only one wash of cold diH2O, you probably haven't. I'd recommend a minimum of two washes/resuspensions before moving on.
2m
Electroporation
Electroporation
1m
1m
Add Amount40 µL -Amount50 µL of cells your ice cold electrocuvette. Make sure to place the cells at the bottom of the gap.

1m
Assuming you have a reasonable amount of DNA in your plasmid sample, you can add betweenAmount1 µL - Amount2 µL of plasmid DNA. If you want to take no chances, Amount5 µL will probably be more than ample. Again, make sure to place at the bottom of the gap, ideally into the cells.

Reasonable DNA amount being somewhere 20+ ng/uL.
1m
Give a gentle tap to the side of the cuvette in order to mix the plasmid and the cells. Return cuvette to ice.
30s
Take the cuvette(s), SOC media, 1000 uL pipette, and a sweetie jar for tip disposal to the electroporation station.
1m
Set the electroporation machine to 2.5 V, 200 U.
1m
Electroporate one cuvette at a time, carefully wiping down the outside to make sure it's dry. Press the two electroporation buttons simultaneous and wait for the sound of the current to go through. As soon as you hear that sound, you can let go of the buttons. You should see a time constant flash up on the screen. Anything between 4-6 seconds as a time constant should not be a cause for alarm.

Note: if you happen to arc your cells (as in, there is a big flash and a bang), you probably didn't get your cell pellet sufficiently clean enough. This happens when there are some salts still in the solution, and your cells are now dead. RIP. If you have more cells left that you have not put in a cuvette, you can take this time to re-wash via ice cold diH2O, spinning, so on. If not, you're back to square one. This is a rare occurrence, so don't panic prematurely.
2m
Remove the cuvette from the machine as soon as possible and immediately add Amount900 µL of SOC media. Keep on the bench. Do not put back on the ice.

If applicable, do the rest of your electroporations, leaving each cuvette with the SOC media at room temperature.
Cell grow-up
Cell grow-up
1h 35m
1h 35m
Once you have done all of your electroporations, use a pipette to remove all of the liquid from the cuvette and put into a fresh sterile eppendorf.
5m
Put your cells in their eppendorfs at Temperature37 °C shaking, for anywhere between Duration00:45:00 and Duration01:30:00 .
45m
Plate Amount100 µL of cells onto the correct selection media plate. Let dry under Bunsen burner.

Spin the remaining volume of cells down on the benchtop centrifuge. Pour off supertant and resuspend in small amount of remaining media. Plate the spun down cells on a selection media plate. This will increase the chance of getting back a transformed colony, if the transformation efficiency seems to be low.
Grow plates DurationOvernight in Temperature37 °C incubator.

45m