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. Treat cells with Buffer and store

Protocol

This is a standard protocol for achieving this result.

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2. Step 2
3. Step 3

Protocol

This is a standard protocol for achieving this result.

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

Protocol

This is a standard protocol for achieving this result.

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

Protocol

This is a standard protocol for achieving this result.

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