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RNase A/T1 Mix

#EN0551	1 ml (2 mg/ml of RNase A and 5000 u/ml of RNase T1)
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EN0551 MSDS (English) MSDS (English-USA) MSDS (German)

 

Features

• Yields a higher RNA degradation than RNase A and RNase T1 separately.
• The RNase A is free of DNase activity. It is not necessary to heat the mix before use.

Description
The RNase A/T1 Mix combines the RNA degradation activity of both RNase A and RNase T1. The RNase A specifically hydrolyzes RNA at C and U residues; RNase T1 specifically hydrolyzes RNA at G residues (1).

Source
RNase A: Bovine pancreas.
RNase T1: E.coli cells with a cloned rntA gene of Aspergillus oryzae.

Applications

• Removal of RNA from DNA preparations (2), see Protocol for Isolation of Plasmid DNA (a mini-preparation).
• Removal of RNA from recombinant protein preparations.
• Ribonuclease protection assays (2).

Quality Control
The absence of endodeoxyribonucleases, exodeoxyribonucleases and proteases confirmed by appropriate quality tests. Functionally tested for degradation of RNA in the plasmid DNA isolation procedure.

Concentration
2 mg/ml (approx. 10000 u/ml (200 Kunitz u/ml)) of RNase A;
5000 u/ml of RNase T1

Unit Definition for RNase A
One unit of the enzyme causes an increase in absorbance of 1.0 at 260 nm when yeast RNA is hydrolyzed at 37°C and pH 5.0.
Fifty units are approximately equivalent to 1 Kunitz unit (3).

Unit Definition for RNase T1
One unit of the enzyme causes an increase in absorbance of 1.0 at 260 nm in 15 min when yeast RNA is hydrolyzed at 37°C and pH 7.5.

Storage Buffer
The enzymes are supplied in: 50 mM Tris-HCl (pH 7.4) and 50% (v/v) glycerol.

Inhibition and Inactivation

• Inhibitors: the most potent inhibitor is a ~50 kDa protein from cytosol of mammalian cells, e.g., RiboLock™ RNase Inhibitor.
  Other inhibitors: uridine 2',3'-cyclic vanadate, 5'-diphosphoadenosine 3'-phosphate and 5'-diphosphoadenosine 2'-phosphate (4), SDS, diethyl pyrocarbonate, 4 M guanidinium thiocyanate plus 0.1 M 2mercaptoethanol and metal ions (MgCl₂ at 100 mM concentration is approx. 40% inhibitory, CaCl₂ at 10 mM is approx. 30% inhibitory, Zn²⁺, Fe²⁺, Cu²⁺ are strong inhibitors), mononucleotides (2'-GMP, 3'-GMP, etc.), guanilyl-2',5'-guanosine is a specific inhibitor (5).
• Inactivated by phenol/chloroform extraction.

Related Products

Proteinase K GeneJET™ Plasmid Miniprep Kit Genomic DNA Purification Kit Water, nuclease-free

References

1. Ausubel, F.M., et al., Current Protocols in Molecular Biology, vol.1, John Wiley & Sons, Inc., Brooklyn, New York, 3.13.1-3.13.3, 1994-2005.
2. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
3. Kunitz, M.A., A spectrophotometric method for the measurement of ribonuclease activity, J. Biol. Chem., 164, 563-568, 1946.
4. Raines, R.T., Ribonuclease A, Chem. Rev., 98, 1045-1065, 1998.
5. Eun, H-M., Enzymology Primer for Recombinant DNA Technology, Academic Press, Inc., 1996.

Updated vasario 05, 2008 15:00