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Materials

Chemicals

  • lysosyme

Lysis buffer

  • 50 mM Tris-HCl pH 7.5
  • 50-200 mM NaCl*
  • 5% glycerol (v/v)
  • 1 mM DTT
  • 1 mM PMSF

*The NaCl concentration used in the lysis buffer depends fully on the application. In case of affinity chromatography on a Ni-column the NaCl concentration is usually 200 mM but when the first purification step is ion exchange chromatography no salt should be added.

Stock solutions

  • 1 mg/ml DNase and in water
  • 100 mM PMSF (phenylmethylsulfonyl fluoride) in isopropanol
  • 1M MgCl2

Procedure

1. Resuspend the cells in chilled lysis buffer in a ratio of 1 g cell wet weight to 1 ml lysis buffer.
  • Add the PMSF (10 µl PMSF (100 mM) per ml of celsuspension) at this point.
2. Add lysosyme to a final concentration of 300 µg/ml and incubate the cell suspension at 4°C for 4 h.
3. Add 5 µl MgCl2 (1 M) and 1 µl DNase solution (1 mg/ml) per ml of cell suspension and incubate the solution at 4°C for 30 min.
4. Remove cell debris by ultracentrifugation at 4°C for 30 min at 45 000 rpm using a 45Ti rotor (Beckman).