Dorman:Preparing electrocompetent cells
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.
- Grow the strain of interest overnight on L
- Add 100 μL of the overnight culture to 10 mL of L in a 250 mL flask.
- Grow this to an OD600 of around 0.5
- 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.
- 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.
- 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.
- Spin again for 10 min to pellet the cells. Put a 1.7 mL tube on ice.
- Resuspend in 1mL of water (I use a cut off p1000 tip for this) and transfer to the chilled tube.
- Spin for 5min at 6000 rpm to pelet.
- Repeat the above two steps two more times.
- After this final spin, resuspend in cells in 200 μL water. These cells are ready to electroporate.
- Mix 1μL of plasmid with 40 μL of cells and electroporate them.
- Add 1 mL of L to the cuvette, and let the cells recover at 37°C for 1 hour.
- Plate 100 μL onto approprate selective media.
- Check your plates. I typically get between 100 and 1000 colonies.
--Dan Stoebel 07:19, 26 September 2007 (EDT)