Electrocompetent E. coli

This protocol was submitted by Matt Kaeberlein.

Required Reagants:

• Ampicillin sensitive E. coli cells.
• 1000 mL 1mM HEPES pH 7
• Super broth
• Glycerol solutions: 20% and 10%
• Ethanol
• Dry ice

Protocol:

1. Grow Amp sensitive cells overnight in 5mL super at 37C
2. Dilute the 5mL culture into 1 L of super broth
3. Incubate on shaker at 37C until O.D. 600 = 0.3-0.5 (about 2-2.5 hours).
4. Pour cells into 4 large centrifuge tubes and put on ice for 30 min.
5. Centrifuge cells at 3000 RPM for 20 min.
6. Pour off supernatant. Resuspend cells in 1 L of cold 1mM HEPES.
7. Centrifuge cells at 3000 RPM for 20 min. Pour off supernatant and resuspend in 1L cold sterile water.
8. Repeat step 7.
9. Centrifuge cells at 3000 RPM for 20 min. Pour off supernatant. Resuspend cells in 500 mL cold sterile water (you can combine them into 2 tubes at this point).
10. Centrifuge cells at 3000 RPM for 20 min. Pour off supernatant. Resuspend cells in 30 mL 20% glycerol.
11. Spin cells at 3000 RPM for 20 min (I use a 45 mL Falcon tube for this spin).
12. Prepare dry ice/ethanol bath. Pre-chill 16-20 1.5 mL eppendorf tubes in the bath.
13. Resuspend cells in 2 mL 10% glycerol.
14. Aliqout 150uL of cells into each eppendorf tube. Keep the tubes floating in the dry ice/ethanol bath and pipet the cells directly into them. Use a P-1000 or a 5 mL pipet (something with a wide opening) to avoid damaging the cells.
15. Place cells at -80 immediately for long term storage.

Tips:

• Before starting this protocol be sure to chill about 3 liters of sterile water and the HEPES solution in the fridge or cold room. I usually put the water in the cold room at the same time I start the overnight culture. Also, make sure the centrifuge and rotor are kept cold. Keeping the cells cold at all times during the washes is essential.
• When resuspending the cells they will be very sticky at first. I use an automatic pipeter with a 25 mL pipet to resuspend. After the second wash the cells should go into solution easily. In fact, you want to avoid jostling the centrifute tubes following the spins to prevent excessive cell loss. I find that it's best to do everything right at the centrifuge itself.