1. Streak from a bacterial stock (e.g., DH5alpha, XL1-Blue) and grow cells on a LB plate over night in
a 37 °C incubator.
2. Pick a single colony and inoculate a 5 ml culture. Grow in a 37 °C shaker (225 rpm)
3. Add 1 ml of the overnight culture to 50 ml LB media.
4. Grow cells until the OD600 reaches 0.5.
5. Chill bacteria on ice and centrifuge at 3000 rpm for 15 minutes at 4 °C.
6. Discard the supernatant, carefully resuspend the cell pellet in 25 ml
sterile, ice-cold 100 mM CaCl2.
7. Leave on ice for 15 minutes.
8. Centrifuge at 3000 rpm for 15 minutes at 4 °C.
9. Discard the supernatant, carefully resuspend the cell pellet in 4 ml
sterile, ice-cold 100 mM CaCl2/15% glycerol.
10. Leave on ice. Aliquot 100-200 ml to sterile
eppendorf tubes. Competent cells can be used immediately, or freeze at -70 °C.