How to make chemically competent BL21 pLysS cells ?
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Answer / Rajani Gupta
Making chemically competent BL21 (DE3) pLysS cells involves a series of steps. Here is an outline:n
1. Overnight culture: Prepare an overnight LB broth culture of BL21 (DE3) pLysS cells by inoculating 5 mL of LB broth with a single colony and incubating at 37°C with shaking.n
2. Harvesting cells: Centrifuge the overnight culture at 4,000 x g for 10 minutes at 4°C, discard the supernatant, and resuspend the pellet in 1 mL of ice-cold sterile water.n
3. Washing steps: Add 5 mL of ice-cold 10% glycerol solution (prepared by mixing equal volumes of glycerol and double-distilled water) and centrifuge at 4,000 x g for 10 minutes at 4°C. Discard the supernatant and resuspend the pellet in 2 mL of ice-cold 10% glycerol solution.n
4. Freezing: Aliquot 50 μL of cell suspension into cryovials and freeze on ice for at least 1 hour before storing at -80°C.n
5. Transformation: Thaw the competent cells on ice, add plasmid DNA, and transfer to a chilled electroporation cuvette. Electroporate using appropriate conditions (usually 2 kV, 200 Ω, and 10 μF with a pulse controller). Immediately resuspend the cells in 1 mL of LB broth and incubate at 37°C for 1 hour before plating on selective media (such as LB agar containing antibiotics).
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