Background What is Ribonuclease A (RNase A) enzyme used for? RNase A is commonly used in the processes of plasmid and genomic DNA purification and protein extraction, in addition to RNA sequence analysis and RNase protection assays. Ribonuclease Pancreatic is a secreted enzyme that belongs to the pancreatic ribonuclease family. RNase A is an endonuclease that cleaves internal phosphodiester RNA bonds on the 3'-side of pyrimidine bases. RNase A prefers poly(C) as a substrate and hydrolyzes 2',3'-cyclic nucleotides. RNase A acts on single stranded and double stranded RNA. RNase A Powder The recombinant Ribonuclease A (RNase A) enzyme is produced from yeast cells with a cloned gene encoding genetically engineered Bovine pancreatic ribonuclease.CAS No.: 9001-99-4.E.C.: 3.1.27.5.Molecular Weight: 13.7 kDa.pI: 9.6.pH range: 6-10; high activity in the pH range of 7.6-8.0.Unit Definition: One unit of the RNase A enzyme causes an increase of absorbance by 1.0 unit at 260 nm when yeast RNA is hydrolyzed at 37°C and pH 5.0. Fifty units are approximately equivalent to 1 Kunitz unit.Specific activity: ≥ 40 Kunitz units/mg of protein.Temperature profile: 15-70°C temperature range recommended; maximum activity at 60°C.Extinction coefficient: E1% = 7.1; 280 nm.Purity: >95% by reducing SDS-PAGE. Each lot of the RNase A (powder and solution) was tested to ensure the absence of protease, endonuclease, exonuclease, DNase, and DNA. No need to heat it before use. DNase is not detected in quality control procedure of incubation 40 µg Proteinase K with 1 ug λ DNA for 6 hours at 37°C. The preparation is considered as DNase free.Formulation: lyophilized powder.Reconstitution: The RNase A enzyme is soluble in water and various buffers (1 mg/mL), yielding a clear colorless solution.Solution preparation: The molecular biology grade recombinant RNase A, lyophilized powder is NOT sterile. It can be used to make molecular biology grade RNase A solution. To prepare a 10 mg/mL RNase A stock solution, dissolve 10 mg of RNase A in 1 mL of Tris-Cl (10 mM, pH 7.5)/NaCl (15 mM).Storage buffer: 10 mM Tris-HCl, 15 mM NaCl, pH 7.5.Recommended concentration of RNase A is 1 to 100 µg/mL depending on the application. For removal of RNA from plasmid preparations use 10 μg/ml working solution and incubate sample for 1 hour at room temperature. For preparation of "blunt ends" of double-stranded cDNA use 100 ng/ml working solution.Activators: Potassium and sodium salts. The enzyme is active under a wide range of reaction conditions. At low salt concentrations (0 to 100 mM NaCl), RNase A cleaves single-stranded and double-stranded RNA as well the RNA strand in RNA-DNA hybrids. However, at NaCl concentrations of 0.3 M or higher, RNase A specifically cleaves single-stranded RNA.Inhibitors: By alkylation of His 12 and His 119. The RNase A has a high affinity to glass surfaces. At neutral pH and high concentrations (>10 mg/ml) the enzyme will precipitate. The enzyme is inhibited by diethyl pyrocarbonate (DEPC), guanidinium salts (4 M GuSCN), β-mercaptoethanol, heavy metals and RNase-inhibitors like RIBOPROTECT (RT33). In order to remove the enzyme from a sample, perform a separation with spin columns or several phenol/chloroform extractions. Shipping: The recombinant RNase A powder is shipped with ice packs. Upon receipt, store it immediately at the temperature recommended below.Stability & Storage:Use a manual defrost freezer and avoid repeated freeze-thaw cycles.24 months from date of receipt if stored at -20 to -70°C.12 months from date of receipt if stored at 2–8°C.12 months after reconstitution if stored at -20 to -70°C. Questions and Answers: Question: Can you provide any RNase A buffer recipe?Answer: Depending on the specific applications of the enzyme, the RNase A buffer recipe may be different. Our recombinant RNase A powder can be dissolved in water or storage buffer: 10 mM Tris-HCl, 15 mM NaCl, pH 7.5. Question: Does the recombinant RNase A enzyme work at room temperature?Answer: The recommended temperature range for proteinase K digestion is 15-70°C; optimum activity at 60°C. Question: How to make RNase A solution from powder?Answer: The most common RNase A stock solution of 10 mg/ml is prepared by dissolving the lyophilized RNase A powder in 10 mM Tris-HCl, 15 mM NaCl, pH 7.5. PBS is also fine. Question: How to prepare RNase A work solution?Answer: Recommended concentration of RNase A is 1 to 100 µg/mL depending on the application. For removal of RNA from plasmid preparations, one uses 10 μg/ml working solution and incubates the sample for 1 hour at room temperature. For preparation of "blunt ends" of double-stranded cDNA, one uses 100 ng/ml working solution. Question: How to inactivate the recombinant RNase A?Answer: It can be completely inactivated by phenol/chloroform extraction. Normally DNA purification can tolerate presence of small amount of the enzyme. Most of RNase A is removed during DNA purification by columns or even ethanol/isopropanol precipitation.
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