- 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.
- Bovine pancreatic RNase A
- 10 mM Tris.Cl, pH 7.5
- Equipment and disposables
- Centrifuge tube (15 ml)
- Boiling water bath
- 10 mg/ml pancreatic RNase
- 10 mM Tris.Cl
Preparation of 10 ml of 10 mg/ml DNase free bovine pancreatic RNase A
Prior to start
Set the water bath for boiling.
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.
- Shake the tube occasionally during incubation in the water bath. This will help to transfer heat to all parts of solution.
- 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.
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.
- 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|