Table of Contents

RNAse treatment

Introduction

RNA is a common contaminant in LPS preparations due to cell rupture and leakage of RNA. Ribose appearing in a hydrolysate (sugar analysis) is most often a sign of contaminating RNA. RNA may be removed by treatment with RNAse or alkali, both followed by purification by gel filtration or dialysis.

Reagents

Full list of chemicals

Procedure

  1. Dissolve the LPS in 0.1 M acetate buffer (pH 5) to 20 mg/mL (an opalescent solution may be obtained) or take solution direct from dialysis.
  2. Add 1 mL of 0.1 M CaCl2 solution per 100 mL.
  3. Add 0.4 mg RNAse per mL LPS solution.
  4. Leave at 37 °C for 3-4 h or in a dialysis bag at 22 °C over night, in either case with 1 drop of CHCl3.
  5. Desalt by gel filtration or dialysis over night.
  6. Freeze-dry.

Comments

  1. Chloroform is added as an antibacterial agent.
  2. If phenol extraction has preceded, it is possible to concentrate the solution and run directly.
  3. The enzyme is normally such a small amount that it can be neglected. If desired it may be removed by conventional methods, e.g. phenol extraction.