Dorman:Preparing electrocompetent cells

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This is a quick and dirty protocol. I use it to make E. coli or Salmonella to which I want to introduce plasmid that I've already cloned. This means that I'm adding a large amount of supercoiled plasmid to the cells, so the efficency doesn't need to be great.

Day 0:

  1. Grow the strain of interest overnight on L

Day 1:

  1. Add 100 μL of the overnight culture to 10 mL of L in a 250 mL flask.
  2. Grow this to an OD600 of around 0.5
  3. While the cells are growing, chill around 20 mL of ddH20. If the centrifuges (both bucket and microcentrifuge) aren't chilled, turn them on now.
  4. Harvest the cells by spinning at 4000 rpm for 10 min at 4°C. From here on out, you want to leave your cells on ice.
  5. Pour off the supernatant, and resuspend in 10 mL water. I resuspend by pipetting- something that we wouldn't do if we were being careful and wanted really high efficency. Leave the cells on ice for at least 20 min- longer is fine.
  6. Spin again for 10 min to pellet the cells. Put a 1.7 mL tube on ice.
  7. Resuspend in 1mL of water (I use a cut off p1000 tip for this) and transfer to the chilled tube.
  8. Spin for 5min at 6000 rpm to pelet.
  9. Repeat the above two steps two more times.
  10. After this final spin, resuspend in cells in 200 μL water. These cells are ready to electroporate.
  11. Mix 1μL of plasmid with 40 μL of cells and electroporate them.
  12. Add 1 mL of L to the cuvette, and let the cells recover at 37°C for 1 hour.
  13. Plate 100 μL onto approprate selective media.

Day 2:

  1. Check your plates. I typically get between 100 and 1000 colonies.

--Dan Stoebel 07:19, 26 September 2007 (EDT)

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