Oneill Lab:Competent Cells

Michael J. O'Neill Lab University of Connecticut Department of Molecular and Cell Biology

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Scott-Simanis Protocol

For making chemically competent E. coli

Materials

• ψb
• ψa plates
  • ψa is made by adding agar to ψb liquid media to a concentration of 1.5% (w/v) and autoclaving
• TFBI
• TFBII

Procedure

1. Streak frozen stock of cells on ψa plate
2. Incubate overnight at 37°C
3. Pick a single colony and inoculate 5ml of ψb
4. Incubate at 37°C overnight
5. Subculture 5ml into 100ml of ψb
6. Grow for about 2 hours, until OD₆₀₀ = 0.5
7. Chill cells on ice for 5 minutes
8. Pellet cells at 4000 rpm for 5 minutes at 4°C
9. Decant media and resuspend cells in 2/5 volume of TFBI
10. Chill cells on ice for 5 minutes
11. Pellet cells at 4000 rpm for 5 minutes at 4°C
12. Decant media and resuspend cells in 1/25 volume of TFBII
13. Aliquot cells and snap freeze
    • Store at -80°C

Links and Contacts

Seth Kasowitz [1]

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