User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2012/03/07

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Objective

Continue Expression of MBP-intein.

Description

  1. After overnight growth, spin cells at 4500rpm at 4°C for 15 minutes.
  2. Resuspend each of the pellets in 1mL of LB broth with glucose(taken from 1L sterile LB with glucose previously made in 2800mL flasks).
  3. Add each of the resuspended pellets with broth to one of the four 2800mL flasks previously made with 1L of sterile LB with glucose.
  4. Add 1mL 100mg/mL ampicillin to each flask.
    • 100mg/mL ampicillin prepared fresh today with 5mL of distilled water and 0.5g of ampicillin, mixed and sterile filtered.
  5. Incubate these four flasks at 37°C at 165rpm until 0.6 OD600 is reached.
  6. Add 1mL 0.1M IPTG to each flask and and continue growth for 2.5hours.
  7. Spin cells at 4500rpm at 4°C for 15 minutes.
  8. Resuspend cells in column buffer (20mM Tris-HCl (pH 7.4), 200mM NaCl, and 1mM EDTA).
  9. Freeze cells with liquid nitrogen.
  10. Store at -80°C.


Notes

  • The OD600 was 1.3 when the cells were induced with IPTG.