Creating electrocompetent cells

Making of competent cells

Step 1 - Overnight Culture Preparation

Step 2 - Preparing Electrocompetent E. coli Cells for Storage.

1. Add 150-200 mL of LB media to the autoclaved 2 L conical (NB/ culture volume must be 1/10 of the total flask volume).
2. Use a 0.5% inoculum from step 1.
3. Incubate at 30°C until OD₆₀₀ ~0.6 (approximately 4 hours).
4. When culture reaches OD₆₀₀ = 0.6, cool to 4°C

Step 3 (after culture OD₆₀₀ ~ 0.6 has been achieved)

1. Aliquot 50 mL of culture into Falcon tubes. Incubate on ice
2. Pre-cool centrifuge to 4°C
3. Centrifuge the tubes for 10 mins at 4°C at 4000 rpm.
4. Decant supernatant.
5. Wash 1: resuspend pellet in 10 mL of 1 mM Hepes buffer (pH 7). Incubate on ice for 5 mins. Gently disturb tube every 30 seconds to facilitate resuspension of the pellet.
6. Centrifuge for 10 mins at 4°C at 4000 rpm
7. Decant supernatant & resuspend pellet in 10 mL of 1 mM Hepes buffer (pH 7). Incubate on ice for 5 minutes. Gently disturb tube every 30 seconds to facilitate resuspension of the pellet.
8. Add two of the Falcon tubes together when cells are fully resuspended (giving two 20 mL cell suspensions). Top each cell suspension up to 50 mL with 1 mM Hepes (pH 7).
9. centrifuge again for 10 mins at 4°C at 4000 rpm.
10. Decant the supernatant & resuspend the pellet in 10 mL Hepes (pH 7). Incubate on ice for 5 minutes.
11. centrifuge again for 10 mins at 4°C at 4000 rpm.
12. Decant the supernatant & resuspend the pellet in 2 mL of Hepes (pH 7). Incubate on ice for 5 minutes.
13. Add glycerol to a final concentration of 12.5% (vol/vol). Mix thoroughly (but GENTLY) & incubate on ice for 5 minutes.
14. Aliquot 50 µL of cells suspension into 500 µL tubes and store at -80^oC.