Team:Paris Bettencourt/Notebook/Wednesday 3th July/Drug Screen

From 2013.igem.org

Competent Cells : BL21 (DE3) dCysI dFpr dydbk

  • We grew 5ml of sD001 cells overnight from a single colony.
  • This 5ml was used to innoculate 500ml of LB broth.
  • Broth was incubated for 1.5h and optical density was read at 0.62 OD600.
  • Cells were alloquoted into 10 falcon tubes (50ml each) and centrifuged at 4000xg for 20 minutes at 4C.
  • Supernatant was removed and cells were resuspended in 200ml of Buffer 1 (4x 50ml) and left on ice for 20 minutes.
  • Cells were centrifuged again for 20 minutes at 4000xg.
  • Supernatant was removed and cells were resuspended in 12ml Buffer 2.
  • Cells were alloquoted at 500ul into microcentifuge tubes and frozen at -80C.
Buffer 1: 50mM CaCl2
Buffer 2: 0.53ml 2M CaCl2 2.8ml 60% Glycerol 8.67ml sterile H2O