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Star Republic: Guide for Biologists

Calcium chloride competent cell

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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) over night.

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.