Oneill Lab:Competent Cells

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

Back to O'Neill Lab Protocols

Scott-Simanis Protocol
For making chemically competent E. coli

Materials

 * &psi;b
 * &psi;a plates
 * &psi;a is made by adding agar to &psi;b liquid media to a concentration of 1.5% (w/v) and autoclaving
 * TFBI
 * TFBII

Procedure

 * 1) Streak frozen stock of cells on &psi;a plate
 * 2) Incubate overnight at 37&deg;C
 * 3) Pick a single colony and inoculate 5ml of &psi;b
 * 4) Incubate at 37&deg;C overnight
 * 5) Subculture 5ml into 100ml of &psi;b
 * 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&deg;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&deg;C
 * 12) Decant media and resuspend cells in 1/25 volume of TFBII
 * 13) Aliquot cells and snap freeze
 * 14) *Store at -80&deg;C

Links and Contacts
Seth Kasowitz [mailto:seth.kasowitz@uconn.edu]

|O'Neill Lab Homepage