Transformation — non-competent E. coli

• Note: There is pre-made sterile CaCl2 on a rack on the chemical shelf in a conical tube. If there isn't, check with Eric. CaCl2 has to be autoclaved before using.

1. Chill an overnight culture of E. coli cells on ice for at least 1 hour.
2. Turn on small water bath to 42 degrees.
3. Transfer 1.0ml of chilled cells into a microcentrifuge tube. Spin for 10 minutes at 4,000 rpm, using the larger, refrigerated centrifuge that you set to 4 degrees.
4. Remove supernatant without disrupting the bacterial pellet.
5. Add 100ul ice cold 100mM CaCl2. Resuspend the cells via stirring with a pipet tip, but not vortexing or pipetting up or down.
6. Add 100ng of plasmid to tube. Mix via stirring, but not vortexing or pipetting up or down.
7. Allow the cells to incubate for 40 minutes on ice.
8. Heat shock at 42 degrees in the small water bath for exactly 45 seconds.
9. Immediately return tubes to ice for at least 2 minutes.
10. Add 900ul LB and grow with shaking for 30 minutes. This will dilute the CaCl2 to a non-competent concentration.
11. Plate 300ul onto antibiotic plates.