Team:Queens Canada/Bacterial-Cell-Lysis

<!DOCTYPE html> Bacterial Cell Lysis · Benchling

Bacterial Cell Lysis

Introduction

Materials

  • Resuspension buffer
    • 20 mM Tris-HCl (pH 7.6)
    • 2 M Urea

Procedure

  1. Harvest cells at 5 000 rpm for 20 minutes.
  1. Resuspend pellets in 25 mL of resuspension buffer.
  1. Sonicate to lyse cells.
  1. Sonicated pellets shoud be centrifuged at 12000 g for 20 min.
  1. After removing supernatant, pellets should be resuspended.
  1. Sonicate cells in 20 mL of Resuspension buffer (20 mM Tris-HCl (pH 7.6) and 6M urea).
  1. Repeat twice.
  1. Keep supernatant after final centrifugation .
  1. Final supernatant was mixed with Ni-NTA agarose pre-equilibrated with resuspension buffer 2.
  1. Incubated for 1 hr at 4C.
  1. Pour entire mixture into column and initial flow through was collected for further analysis.
  1. Wash column with 5mL of wash buffer (20 mM Tris-HCl (pH 7.6), 6 M Urea, 20 mM imidazole) three times.
  1. Elute protein of interest with 1.5 mL of elution buffer (20 m Tris-HCl (pH 7.6), 6 M Urea, 0.3 M imidazole).
  1. Elution fraction was dialyzed against 1L of Dialysis Buffer (30 mM Tris-HCl (pH 7.6), 10 mM EDTA) at 4C overnight.