Calcium Chloride Competent Cells

This is a common protocol to make semi-competent E. coli cells (DH5alphas, BL21(DE3), etc.). Inoue method gives better-quality cells, but is also more work.

Contents

1. Preparation
2. Day 1
3. Day 2
4. Day 3
5. Day 4
6. Reference

1. Preparation

• Make 1L of 0.1 M CaCl₂ stored at 4°C.
• Sterile 80% glycerol

2. Day 1

1. Streak out the desired cell line on an LB plate using a sterile toothpick or tip. Grow overnight at 37°C.

3. Day 2

1. Make four 2L flasks, each filled with 250 mL autoclaved LB.
2. Take one colony from the plate and inoculate it into 10 mL LB. Grow overnight.

4. Day 3

1. Pipet 2 mL of overnight culture into each of the flasks.

2. Grow at 37°C in shaking incubator until OD₆₀₀ = 0.5 (takes approximately 2 hours).

3. Pour two flasks into a 1L centrifuge bottle, repeat for other two flasks.

4. Centrifuge at 4°C, 5000 RPM for 10 minutes. Did you notice how it said RPM and not RCF? Great!

5. Move to the cold room for the rest of the protocol.

6. CAREFULLY decant supernatant.

7. Resuspend bacterial pellets with 100 mL cold 0.1M CaCl₂ (100 mL in each centrifuge bottle).

   Tip

   To respuspend, use a cold, sterile, 25ml serological pipette tip and pipet back and forth until chunks are gone. DO NOT VORTEX.

8. Add 50 ml of each bottle to a 50 ml conical (4 tubes total).

9. Centrifuge in the swinging bucket rotor at 4°C, 5300 RPM for 15 minutes.

10. Carefully decant again and resuspend each tube using 50 mL of 0.1M CaCl₂.

11. Incubate the resuspended cells on ice for 4 hours.

    1. Chill 0.7 mL snap tubes, a couple tube racks, the repeat pipettor, several 2.5 mL tips and 80% glycerol.
    2. Just before incubation ends, put snap tubes with lids open in some tube racks. This way you can quickly fill them up with a repeat pipettor, close them, and throw them in liquid nitrogen.

12. Centrifuge in the swinging bucket rotor at 4°C, 5300 RPM for 15 minutes.

13. Resuspend with 20 mL TOTAL 0.1M CaCl₂ (put 20 mL in one conical tube, resuspend, then transfer to another conical and repeat).

14. Put the falcon tube on ice while you work with it.

15. Add 6.25 mL 80% glycerol to resuspension mix and pipet back and forth to mix.

16. Make 50 µL aliquots of cells and flash freeze in liquid nitrogen.

17. Store aliquots at -80°C.

5. Day 4

Make sure to test the transformation efficiency. https://www.neb.com/faqs/1/01/01/how-should-i-calculate-the-transformation-efficiency-c3019 PDF

• If you would like a standardized measure of efficiency, use a Puc19 plasmid.
• Make sure to test the downstream application of the cell as well (ie DNA/Protein production)

6. Reference

• CSH Protocols