Objectives

The objective here is to make a stock of E.Coli DH5⍺ competent cells for subsequent transformations.

Materials

Stock concentrations:

O/N DH5⍺ pre-culture: O/N inoculation of 100 µL DH5⍺ into 100 mL LB.

0.1M CaCl2: prepared the 05/06

0.1M CaCl2/15% Glycerol: prepared the 05/06

Protocol

Competence:

1. Inoculation of 3 mL O/N culture in 100 mL LB in 500 mL erlenmeyer.

2. Incubation at 250 rpm at 37°C until the DO 0,6 —> DO = 0.615

3. Cells were transferred to 3 sterile ice-cold 50 mL Falcon tubes. 20 mL in each falcon tube.

4. Incubate on ice for 30 min. Do not allow cells to warm up over 4°C.

5. Spin cells at 4000 rpm for 10 min at 4°C.

6. Discard supernatant and try to drain all remaining media.

7. Gently resuspend on 10 mL cold 0.1M CaCl2

8. Incubate on ice 20 min

9. Centrifuge 10 min at 4,000 rpm at 4°C

10. Discard supernatant and gently resuspend on 5 mL cold 0.1M CaCl2/25% Glycerol

11. Transfer in 1.5 mL eppendorf (100 µL/tube)

12. Store at -80°C

NB: The competency of the prepared cells will be tested the 10/06.