Team:Queens Canada/Ecoli

Cell Lysates from E. Coli · Benchling

Cell Lysates from E. Coli

Introduction

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Materials

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

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 at this point
  1. Add 5 uL MgCl2 (1M) and 1 uL DNase solution (1 mg/mL) per mL of cell suspension and incubate the solution at 4C for 30 min.
  1. Remove cell debris by ultracentrifugation at 4C for 30 min at 45 000 rpm.