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Protein Expression

Source: Contributed by Mark's Lab
Date Added: Tue May 14 2002
Date Modified: Thu Apr 29 2004



Note: If glycerol cultures of transformed cells are available go directly to day2.

  1. Make competent cells
  2. Transform competent cells


  1. Put 5 ml filter sterile LB into universal tube plus appropriate antibiotics
  2. Inoculate 5mL culture with  colony picked from plate or scratched off glycerol culture
    Grow cells during the day
  3. Prepare 100mL sterile filtered LB  in 0.5L shaker flask and autoclave
    Add appropriate antibiotic before inoculation
    Take 1 mL from day culture to inoculate 100ml LB night culture

Things to have ready before starting:


  1. Harvest cells from night culture in autoclaved 0.5L buckets
  2. Centrifuge in GS3 rotor 10-15' @ 5000 rpm
  3. Resuspend cell pellet gently in 10 mL LB (use vortex, avoid too much pipetting)
  4. Inoculate 0.5L of expression medium with 1-2 mL of cell suspension
  5. Grow cells in shakers at appropriate temperature and speed
    (usually 37C and 200 rpm, make sure temperature is really there)
    Monitor growth by measuring OD @ 600nm (in steps of ~1h)
  6. Induce expression between 0.7-1.0 OD with 1/1000 0.75 M IPTG
    (0.89 g/ 5 mL fresh stock, sterile filtered)
  7. Continue growth for another 3-5h
  8. Harvest cultures in 0.5L buckets
  9. Centrifuge 15' @ 5000 rpm in GS3 rotor, discard supernatant
  10. Take up cells in 6 mL per L medium appropriate buffer
  11. Freeze cell suspension in -20 freezer

Note: it is also possible to freeze the cell pellets in the 0.5L buckets after removing the medium

Things to have ready before starting:

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