Team:Duke/Notebook/Protocols

Chemically Competent Cells

This protocol for making chemically competent E. coli for transformations. It is derived from the official iGEM protocol, which can be found here

1. Making CCMB 80 Buffer
   • 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)
   • 80 mM CaCl2.2H2O (11.8 g/L)
   • 20 mM MnCl2.4H2O (4.0 g/L)
   • 10 mM MgCl2.6H2O (2.0 g/L)
   • 10% glycerol (100 ml/L)
   • adjust pH DOWN to 6.4 with 0.1M HCl if necessary
2. Culturing Cells
   • Scrape cells from a colony or frozen stock of the desired strain
   • Inoculate into 5 mL SOC (or LB+Antibiotic for plasmid-containing strains)
   • Grow for ~8 hrs (morning to late afternoon) in 37C shaker
   • Add 5 mL of culture to 250 mL SOC (or LB+Antibiotic) in a shaker flask
     • 250 mL culture will yield approximately 50 chemically competent samples. For smaller batches, we add 1 mL into 50 mL.
   • Grow overnight for ~16 hrs in a shaker at room temperature
3. Treating cells
• Transfer culture into 50 mL centrifuge tubes
• Pellet cells at 4500 RPM for 10 mins
• Pour off supernatant and resuspend cells in 40 mL CCMB 80 Buffer
• Incubate on ice for 20 minutes
• Pellet cells at 4500 RPM for 10 mins
• Pour off supernatant and resuspend cells in 5 mL CCMB 80 Buffer
• Incubate on ice for 20 minutes
• Aliquot 750 uL each into pre-chilled microcentrifuge tubes
• Store at -80C until use
• Note: we never refreeze competent cells once thawed. Any unused cells in an aliquot are discarded.

Protocol

This is a standard protocol for achieving this result.

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Protocol

This is a standard protocol for achieving this result.

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Protocol

This is a standard protocol for achieving this result.

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Protocol

This is a standard protocol for achieving this result.

1. Step 1
2. Step 2
3. Step 3