Chemical transformation

Preparation of chemically competent cells

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.

Electroporation

Preparation of electrocompetent cells

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 a