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