Have the following solutions at 0-4 deg C:
a) 100 mM MgCl2
b) 100 mM CaCl2-15% glycerol
c) sterile GSA bottles and pre-cooled rotor
1. Grow a 5 mL overnight culture of bacteria.
2. Dilute 1:100 and shake at 37 deg C (5 mL into 500 mL).
3. After 1.5-2 hours, A600= 0.5-0.6 (0.4 also works well).
4. When cells reach proper density, transfer to GSA bottles and spin down 5000 rpm (JA-10), 10 minutes at 4 deg C.
5. Pour off supernatant and keep the cells on ice.
6. Resuspend in 100 mL of 100 mM MgCl2 (ice-cold) by pipetting up and down.
7. Incubate on ice 20-30 minutes.
8. Spin down cells at 4000 rpm for 10 minutes at 4 deg C. Discard supernatant and keep cells on ice.
9. Cool small eppendorf tubes on ice.
10. Resuspend in 10 mL of 100 mM CaCl2-15% glycerol (ice-cold).
11. Aliquot into tubes (170 µl/tube) and put at -80 deg C (quick freezing not necessary).
Transformation of frozen competent cells
1. Thaw frozen cells on ice, 10-15 minutes.
2. Add 80 µl cells to the 20 µl ligation reaction.
3. Incubate on ice for 5 minutes.
4. Heat shock cells for 10 minutes at 37 deg C.
5. Bring up in 1 mL LB and shake gently for 1 hour at 37 deg C.
1. Inoculate 1 L of LB containing 1/2 the amount of NaCl as normal with 5 ml of an overnight culture. Shake at 37 deg C until mid log OD(600) = 0.5. Incubate cells in ice water for 15 minutes.
2. 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.
3. Pour 500 ml of cells into each of the pre-chilled centrifuge bottles. Pellet cells at 6000xg for 15 minutes at 4 deg C.
4. 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.
5. Pellet cells at 6000xg for 15 minutes at 4 deg C.
6. 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.
7. Pellet cells at 600g for 15 minutes at 4 deg C.
8. Carefully pour off the glycerol and resuspend the cells up to 2 ml using 10% glycerol.
9. Distribute cells in 80 µl aliquots into microfuge tubes and quick freeze in a -70 deg C bath (liquid N2 is okay, too).
Transformation of electrocompetent cells
1. Chill cuvettes on ice for 5 minutes. Thaw cells on ice.
2. 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.
3. Transfer the DNA and the cells into a cuvette. Keep on ice.
4. Set the electroporator to 2.5 kV, 25 µF, and 400 ohms.
5. Pulse, and record the actual voltage and time constant.
6. 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 microfuge tube.
7. Incubate 30-60 minutes with moderate shaking at 37 deg C.
8. Plate aliquots on LB plates containing the appropriate antibiotics.
Last Modified: Aug 29, 2001