- 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 5 – 10 minutes usually eliminate 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.
- Plasmid isolation
- Protocol – Plasmid isolation by alkaline lysis method (miniprep)
- Protocol – Plasmid isolation by boiling method (miniprep)
- Preparation of agarose gel for DNA analysis
- Equipment and disposables
- 15-ml screw-cap graduated polypropylene centrifuge tube
- Boiling water bath
- 10 mg/ml pancreatic RNase
- 10 mM Tris.Cl, pH 7.5
Preparation of 10 ml of 10 mg/ml DNase free bovine pancreatic RNase A
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 screw-cap graduated polypropylene centrifuge tube. Add 7 ml Milli Q/Deionized water and 100 µl of 1M Tris.Cl (pH 7.5). Mix by inverting the tube several times until all RNase A is dissolved completely.
We recommend to use disposable DNases-free 15-ml screw-cap graduated polypropylene centrifuge tube. Since these tubes have milliliter marks, you can adjust the solution volume without transferring the solution to measuring cylinder. These tubes are heat resistant, inert, and can be centrifuged to remove any precipitate from the solution.
- Avoid frothing while mixing the ingredients.
Step 2: Adjust the volume to 10 ml with Milli Q/Deionized water.
- We recommend you to give a short spin to 15 ml tube just before adjusting the volume. Short spin will help to collect all liquid drops which are adhered to sides and lid of the tube.
Step 3: Keep the centrifuge tube on boiling water bath for 5 – 10 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 at -20°C. RNases A is very stable and can be stored at -20°C/-80°C for 1 year.
- 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|
|1M Tris.Cl (pH 7.5)||50 µl||100 µl||500 µl||1000 µl|
- Birnboim, H.C. (1983). A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 1983;100:243-55.
- RNase solution. Cold Spring Harbor Protocols, pdb.rec8818.