====== RNAse treatment ======

===== Introduction =====

RNA is a common contaminant in LPS preparations due to cell rupture and leakage of RNA. Ribose appearing in a hydrolysate ([[sop1|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 ====

  * Ribonuclease A
  * 0.1 M CaCl<sub>2</sub> or MgCl<sub>2</sub>
  * 0.1 M Sodium acetate buffer

[[chemicals|Full list of chemicals]]

==== Procedure ====

  - 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.
  - Add 1 mL of 0.1 M CaCl<sub>2</sub> solution per 100 mL.
  - Add 0.4 mg RNAse per mL LPS solution.
  - 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 CHCl<sub>3</sub>.
  - Desalt by gel filtration or dialysis over night.
  - Freeze-dry.

==== Comments ====

  - Chloroform is added as an antibacterial agent.
  - If phenol extraction has preceded, it is possible to concentrate the solution and run directly.
  - The enzyme is normally such a small amount that it can be neglected. If desired it may be removed by conventional methods, //e.g.// [[sop301|phenol extraction]].