D. Make competent E.coli cells

What are competent cells?

Competent E.coli cells are more likely to incorporate foreign DNA if their cell walls are made more permeable so that DNA can pass through easily. Bacterial cells are naturally competent to a certain extent. They can be made artificially more competent in a laboratory when treated with calcium chloride (CaCl2) to make them transiently more permeable. The CaCl2 creates pores in the bacterial cell wall. The DNA is then forced into the cell using heat shock (at 42°C). This is the process of transformation.

If you perform a lot of transformations, making your own competent cells is more cost effective. The process is relatively simple and the cells can be made in bulk and snap frozen for later use. If, however you need a high efficiency of transformation, for example, if you are working with a low concentration of plasmid, you should purchase competent cells.

There are various methods and kits that can be used to make competent cells. In the following protocol, you will make E.coli cells competent using CaCl2. Adding CaCl2 to the cell suspension promotes the binding of the plasmid DNA to lipopolysaccharides in the inner core of the outer membrane of Gram negative bacteria, such as E.coli. Upon subsequent heat shock, the bound plasmid DNA can pass into the cell.

  1. Place 4 x 250 mL centrifuge bottles on ice.
  2. Split the 1L of bacterial culture into the ice cold centrifuge tubes.
  3. Centrifuge at 3000 x g (~4000 rpm) in a Beckman JA-10 rotor for 15 minutes at 4°C.
  4. Decant the supernatant and resuspend the bacterial pellet in 100 mL of ice cold MgCl2.
  5. Combine the suspensions into one bottle and prepare a balance bottle.
  6. Harvest the cells by centrifugation at 2000 x g (~3000 rpm) in a Beckman JA-10 rotor for 15 minutes at 4°C.
  7. Decant the supernatant and resuspend the pellet in 200 mL of ice cold CaCl2. Keep this cell suspension on ice for 20 minutes.
  8. Place 1.5 mL microfuge tubes on ice.
  9. Harvest the cells by centrifugation at 2000 x g (~3000 rpm) in a Beckman JA-10 rotor for 15 minutes at 4°C.
  10. Rinse a 50 mL conical tube with ddH2O and cool on ice.
  11. Decant the supernatant and resuspend the pellet in 50 mL of ice cold 85 mm CaCl2, 15% glycerol.
  12. Transfer the suspension to the 50mL conical tube.