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Competent Cell Production

Day 1

1. Streak out frozen glycerol stock of bacterial cells (Top 10, DH5α, etc.) onto an LB plate without antibiotics.
2. Grow plate overnight at 37°C.

Day 2

1. Autoclave:
   • 2 L of ddH₂O
   • 100 mL of 10% v/v glycerol (molecular biology grade)
   • 1 L of LB (or preferred media)
   • 4 centrifuge bottles and caps
   • lots of microfuge tubes
2. Chill overnight at 4°C:
   • ddH₂O
   • 10% glycerol
   • centrifuge rotor
3. Prepare starter culture of cells.
   • Select a single colony of E. coli from fresh LB plate and inoculate a 10 mL starter culture of LB (or preferred media).
   • Grow culture at 37°C in shaker overnight.
   • Possible media substitutes include SOB, 2xYT, etc.
   • All glassware should be detergent-free, as trace detergent residue reduces competency.

Day 3

1. Inoculate 1 L of LB media with 10 mL starter culture and grow in a 37°C shaker. Measure the OD₆₀₀ every hour, then every 15 - 20 minutes when the OD gets above 0.2.
2. When the OD₆₀₀ reaches 0.35 - 0.4, immediately put the cells on ice. Chill the culture for 20 - 30 minutes, swirling occasionally to ensure even cooling. Place centrifuge bottles on ice at this time.
   • It is important not to let the OD get any higher than 0.4. It usually takes about 3 hours to reach an OD of 0.35 when using a 10 mL starter culture.
   • It is also very important to keep the cells at 4°C for the remainder of the procedure. The cells, and any bottles or solutions that they come into contact with, must be pre-chilled to 4°C.
3. (SPIN #1) Split the 1 L culture into four parts by pouring about 250 mL into ice-cold centrifuge bottles. Harvest the cells by centrifugation at 1000g (~2400 rpm in the Beckman JA-10 rotor) for 20 minutes at 4°C.
4. Decant the supernatant and resuspend each pellet in 200 mL of ice-cold ddH₂O.
5. (SPIN #2) Harvest the cells by centrifugation at 1000g (~2400 rpm in the Beckman JA-10 rotor) for 20 minutes at 4°C.
6. Decant the supernatant and resuspend each pellet in 100 mL of ice-cold ddH₂O.
7. (SPIN #3) Combine resuspensions into 2 centrifuge bottles (so each contains about 200 mL of cell suspension). Harvest the cells by centrifugation at 1000g (~2400 rpm in the Beckman JA-10 rotor) for 20 minutes at 4°C. At this step, rinse two 50 mL conical tubes with ddH₂O and chill on ice.
8. Decant the supernatant and resuspend each pellet in 40 mL of ice-cold 10% glycerol. Transfer each suspension to a 50 mL conical tube.
9. Harvest the cells by centrifugation at 1000g (~2100 rpm in the Beckman GH-3.8 rotor) for 20 minutes at 4°C. Start putting 1.5 mL microfuge tubes on ice if not already chilled.
10. Carefully aspirate the supernatant with a sterile Pasteur pipette (pellets lose adherence in 10% glycerol). Resuspend each pellet in 1 mL of ice-cold 10% glycerol by gently swirling. The final OD₆₀₀ of the resuspended cells should be ~ 200 - 250.
11. Aliquot into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. Store frozen cells in the -80°C freezer.