Protocol Code: CT4

Fast CaCl₂ Combined Competency and Transformation

Required Solutions

• 2.5mL of 50mM CaCl₂
• 5.5 mL liquid LB media

Competency

1. Take cell culture and grow in 5mL LB liquid media overnight until OD₆₀₀ = 0.6-0.7.
2. Cool cells on ice for 10 minutes.
3. Centrifuge cells at 3000rpm, 4^oC, for 5 minutes.
4. Discard supernatant and place cells back on ice.
5. Resuspend cells in 2mL of cold CaCl₂ (50mM).
6. Cool cells on ice for 15 minutes.
7. Centrifuge cells at 3000rpm, 4^oC, for 5 minutes.
8. Discard supernatant and place cells back on ice.
9. Resuspend cells in 500µL of cold CaCl₂ (50mM).

Transformation

1. Take 100-200µL of the resuspended cells and place in a precooled microfugetube.
2. Add DNA ligation mixture.
3. Incubate on ice for 20-30 minutes. Prepare 42^oC water bath.
4. Heat shock cells in water bath for 2 minutes.
5. Move cells back onto the ice.
6. Add 0.5mL LB media.
7. Incubate cells at 37^oC for 1 hour.
8. Spread cells over 1-2 LB/antibiotic plates, and invert in incubator overnight.