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Creating electrocompetent cells

Making of competent cells

Step 1 - Overnight Culture Preparation

• 1. Transfer 10 mL of sterile LB to a 50 mL Falcon Tube.
• 2. Inoculate media with a single colony of E. coli cells.
• 3. Incubate overnight at 37°C, in a shaker at 230 rpm.

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.

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