Preparation of chemically competent cells
- Have the following solutions at 0-4 °C:
Grow a 5 mL overnight culture of bacteria.Dilute 1:100 and shake at 37 °C (5 mL into 500 mL).After 1.5-2 hours, A600= 0.5-0.6 (0.4 also works well).When cells reach proper density, transfer to GSA bottles and spin down 5000
rpm (JA-10), 10 minutes at 4 °C.Pour off supernatant and keep the cells on ice.Resuspend in 100 mL of 100 mM MgCl2 (ice-cold) by pipetting up and down.Incubate on ice 20-30 minutes.Spin down cells at 4000 rpm for 10 minutes at 4 °C. Discard supernatant
and keep cells on ice.Cool small eppendorf tubes on ice.Resuspend in 10 mL of 100 mM CaCl2-15% glycerol (ice-cold).Aliquot into tubes (170 µl/tube) and put at -80 °C (quick freezing not
- 100 mM MgCl2
- 100 mM CaCl2-15% glycerol
- sterile GSA bottles and pre-cooled rotor
Transformation of frozen competent cells
- Thaw frozen cells on ice, 10-15 minutes.
- Add 80 µl cells to the 20 µl ligation reaction.
- Incubate on ice for 5 minutes.
- Heat shock cells for 10 minutes at 37 °C.
- Bring up in 1 mL LB and shake gently for 1 hour at 37 °C.
Preparation of electrocompetent cells
- Inoculate 1 L of LB containing 1/2 the amount of NaCl as normal with 5 ml of
an overnight culture. Shake at 37 °C until mid log OD(600) = 0.5. Incubate
cells in ice water for 15 minutes.
- Meanwhile, incubate 2 cetrifuge bottles, 12 mL of 10% glycerol, 500 ml of sterile
water, and a 50 ml conical tube on ice for at least 30 minutes.
- Pour 500 ml of cells into each of the pre-chilled centrifuge bottles. Pellet cells at 6000xg for 15 minutes at 4 °C.
- Pour off the supernatant and resuspend each pellet in 250 ml of ice-cold sterile water. Resuspend the cells while on ice by loosening the pellet by scraping with
a sterile pipet, quickly vortexing for a few seconds and then shaking the bottles
in a circular motion by hand until the cells are completely resuspended. Maintain
cells on ice or ice water at all times.
- Pellet cells at 6000xg for 15 minutes at 4 °C.
- Pour off the water and resuspend each pellet ion 2.5 ml of ice cold 10% glycerol.
This can be accomplished by breaking the pellet with a cold pipet followed by
tituration (sucking in and out of the pipet). Combine cells and transfer to an
ice cold 50 ml conical tube.
- Pellet cells at 600g for 15 minutes at 4 °C.
- Carefully pour off the glycerol and resuspend the cells up to 2 ml using 10%
- Distribute cells in 80 µl aliquots into microfuge tubes and quick freeze in
a -70 °C bath (liquid N2 is okay, too).
Transformation of electrocompetent cells
- Chill cuvettes on ice for 5 minutes. Thaw cells on ice.
- Add 5 pg - 5 µg plasmid DNA in 1 µl to the cells. Mix by tapping the tube or
swirling the contents with a pipet tip.
- Transfer the DNA and the cells into a cuvette. Keep on ice.
- Set the electroporator to 2.5 kV, 25 µF, and 400 ohms.
- Pulse, and record the actual voltage and time constant.
- Immediately add 1 ml SOC medium (0.5% yeast extract, 2% tryptone, 10 mM NaCl,
2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose) and transfer to a sterile
- Incubate 30-60 minutes with moderate shaking at 37 °C.
- Plate aliquots on LB plates containing the appropriate antibiotics.