How to prepare electrocompetent ''E. coli''

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Preparing Electrocompetent Cells

  1. Mix 1ml overnight culture with 50ml LB form Falcon tube in Erlenmeyer flask; put 200$\mu$l in 96-well plate; incubate at 37°C.
  2. Track the $OD_{600}$ to $0,5-0,6$; immediately cool down tubes with ice; chill for 10min; \textbf{keep cells cold} from now on.
  3. Wash cells by spinning them down at 4000 x g, 18min, 4°C; discard supernatant; resuspend with 35ml dH2O.
  4. Again wash cells by spinning them down at 4000 x g, 18min, 4°C; discard supernatant; resuspend with 35ml dH2O.
  5. Spin cells down; discard supernatant; resuspend with 2ml 10$\%$ glycerol; transfer to eppi.
  6. Spin 2min at topspeed and 4°C on table centrifuge; discard supernatant; resuspend in 1ml 10$\%$glycerol.
  7. Tansfer 70$\mu$l in 14 PCR tubes (or eppis) respectively; use directly for Electroporation (keep on ice!)
  8. Or freeze at $-$80°C


This protocol was created after reviewing several protocols. The reviewed protocols were obtained from

http://www.its.caltech.edu/~bjorker/Prep_of_electocomp_cells.pdf
Molecular Cloning: A Laboratory Manual by Joseph Sambrook, David William Russell, CSHL Press
Protocols from other groups at IST Austria