78,00 €The Ribonuclease A (RNase A) is a 13.7 kDa (monomer) endoribonuclease isolated from bovine pancreas, which selectively cleaves single-stranded RNA 3’ next to pyrimidine residues (cytosine, uracil). It degrades RNA to cyclic nucleotide monophosphates to 5’- OH and 2’-, 3’-cyclic monophosphate. The enzyme exhibits no endonuclease or exonuclease activity towards DNA substrates. The RNase A is used to remove RNA during the isolation procedures of plasmid and genomic DNA.
The enzyme is very active under a wide range of reaction conditions and difficult to inactivate. At low salt concentrations (up to 100 mM NaCl), the RNase A cleaves single- and double-stranded RNA as well as an RNA strand in RNA-DNA hybrids. However, under high salt concentrations (>300 mM NaCl), the RNase A specifically cleaves single-stranded RNA.
- Enzyme activity: > 80 Kunitz units/mg protein
- Supplied in a salt-free, freeze dried form
- Purification of RNA-free DNA
- Plasmid and genomic DNA isolation
- Removal of RNA during recombinant proteins preparations
- RNA protection assays
- Mapping of single-base mutations in DNA or RNA
One unit of activity is defined as that amount of enzyme which causes the hydrolys is of RNA to yield a velocity constant, k=1, at 25°C and pH 5.0 (Kunitz-Unit).
Protocol & Manual
RNase A (RP145) - Manual
Stock solutions should be prepared to a final concentration 1 – 10 mg/ml by resuspending in 10 mM Tris-HCl (pH 7.5), 15 mM NaCl, 50% (v/v) glycerol or in TE buffer.
The recommended working solution concentration depends on 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.
Keep at -20°C (lyophilized or in a 50% glycerol solution) for long-term storage or at +4°C for up to several weeks. When stored at -20°C (lyophilized or in a glycerol solution), the enzyme remains stable for several years.
Shipping at ambient temperature.
- The RNase A has a high affinity to glass surfaces.
- At neutral pH & 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 Hu (RT35).
- In order to remove the enzyme from a sample, perform a separation with spin columns or several phenol/chloroform extractions.
Quality & Safety
The activity of the enzyme, absence of deoxyribonucleases and proteases has been confirmed in appropriate quality tests.