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Ribonuclease A- DNase-free is a DNase and Protease-free enzyme that can be used directly for RNA digests. Elimination of potential DNAse activity is not necessary. Potential DNAse activities have been removed.
Ribonuclease A is an endo-ribonuclease specifically cleaving single stranded RNA at the 3' side of pyrimidine bases (cytosine and uracil). RNAse A ist used to prepare RNA-free DNA, to digest non-hybridised regions of RNA-DNA hybrids and as a molecular weight marker. The pH optimum for RNAse A is between 7.0-7.5. RNAse A is inhibited by Diethylpyrocarbonate (DEPC) >, guanidinium salt (4M GuaSCN), beta-Mercaptoethanol, heavy metals, vanadyl-ribonucleoside complexes, RNAse-inhibitor from human placenta and by competitive DNA. RNAse A cleaves single and douple stranded RNA and RNA in RNA:DNA hybrids at low salt conditions (100mM NaCl). At high salt conditions (>300 mM NaCl), RNAse A cleaves single-stranded RNA only. The enzyme can only be removed by Proteinase K digest and subsequent phenol extraction.
Stock solution: Stock solutions are prepared at concentrations from 1 - 10mg/mL in 10mM Tris/HCl, pH7.5; 15mM NaCl or in 10mM Tris/HCl, pH7.5; 1mM EDTA, pH8.0 (TE buffer).
The recommended working concentration is 10µg/mL (removal of RNA from plasmid preparations; 1hr, RT) or 100ng/mL (preparation of 'blunt ends' of double-stranded cDNA).
Stability:RNase A aggregates during lyophilizing and storage. It has a high affinity to glas surfaces, which has to be taken into consideration. At neutral pH (e. g. in PBS pH 7.4) and high concentrations (>10mg/mL) the enzyme will precipitate. At +2°C to +8°C (lyophilized) it is stable for several years (dry storage), in solution (-20°C) several years or (+2°C to +8°C) several weeks.
Activity: min. 80 kunits/mg
DNases: not detectable
Proteases: not detectable
MW = approx. 13,700 Da
Product is isolated from BSE free source.
Optimal temperature: 60°C (activity range of 15–70°C); Optimal pH: 7.6 (activity range of 6–10); Inhibitors: ribonuclease inhibitor. Activators of RNase A: potassium and sodium salts.
application:RNase protection assays Remove unspecifically bound RNA Analysis of RNA sequences Hydrolyze RNA contained in protein samples Purification of DNA Pancreatic RNase A specifically cleaves at the 3'-side of pyrimidine (uracil or cytosine) phosphate bonds
Unit Definition:1 unit corresponds to the amount of enzyme which hydrolyzes RNA at a rate constant k = 1 at 25°C and pH 5.0 (Kunitz-units), M. Kunitz, J. Biol. Chem. 164, 563 (1946).
Sicherheits Hinweise / Safety
Klassifizierungen / ClassificationEC-Nr: 232-646-6
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Ieva Bruzauskaite, Jovile Raudoniute, Jaroslav Denkovskij, Edvardas Bagdonas, Sandra Meidute-Abaraviciene, Vaida Simonyte, Daiva Bironaite, Almantas Siaurys, Eiva Bernotiene, Ruta Aldonyte
Cytotechnology. 2017 Feb; 69(1): 1–17. Published online 2016 Dec 1. doi: 10.1007/s10616-016-0021-z