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.

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.

1. Grow this to an OD₆₀₀ of around 0.5

1. 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.

1. 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 for the rest of the time.

1. 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.

1. Spin again for 10 min to pellet the cells. Put a 1.7 mL tube on ice.

1. Resuspend in 1mL of water (I use a cut off p1000 tip for this) and transfer to the chilled tube.