Preparation of DNase-free Bovine pancreatic RNase A

Overview

  • Bovine pancreatic RNase (RNase A) is an endoribonuclease. It specifically degrades single-stranded RNA at C and U residues at higher salt concentrations (0.3 M or higher NaCl concentrations).
  • It cleaves single-stranded as well as double-stranded RNA and also RNA strand in RNA-DNA hybrids at low salt concentrations (0 to 100 mM NaCl).
  • It is active under a wide range of reaction conditions.
  • Unlike DNases, RNase A is quite stable to heat. Boiling RNase A solution for 15 – 20 minutes usually eliminate most of its DNases activity without compromising it’s RNases A activity. This preparation of RNases A is sufficient for most molecular biology work including the preparation of RNA free plasmid DNA.

Requirement  

  • Reagents
  • Equipment and disposables
    • Centrifuge tube (15 ml)
    • Boiling water bath

Composition

  • 10 mg/ml pancreatic RNase
  • 10 mM Tris.Cl

Objective

Preparation of 10 ml of 10 mg/ml DNase free bovine pancreatic RNase A

Prior to start

Set the water bath for boiling.

Protocol

Step 1: To prepare, 10 ml of bovine pancreatic RNase A solution, weigh out 100 mg bovine pancreatic RNase A and transfer it to 15 ml centrifuge tube. Dissolve in 8 ml of 10 mM Tris.Cl (pH 7.5) by inverting the tubes several times.

Step 2: Adjust the volume 10 ml with 10 mM Tris.Cl (pH 7.5).

Step 3: Keep the centrifuge tube on boiling water bath for 15 – 20 minutes.

Tips:
  • Shake the tube occasionally during incubation in the water bath. This will help to transfer heat to all parts of solution.
Precautions:
  • Don’t directly boil the RNase solution on the hot plate. This may lead to precipitation of proteins.

Step 4: Cool the solution to room temperature. Centrifuge at high speed (5,000 rpm) to remove the precipitate.

Storage

Make 1 ml aliquots and store it at -20°C. RNases A is very stable and can be stored at -20°C/-80°C for many years.

Application

  • Plasmid DNA isolation
  • Preparation of RNA-free DNA
Reagents / Volume 5 ml 10 ml 50 ml 100 ml
Pancreatic RNase 50 mg 100 mg 500 mg 1000 mg
10 mM Tris.Cl 5 ml 10 ml 50 ml 100 ml