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RNase T1

#EN0541	100,000 u (1000 u/µl)
#EN0542	500,000 u (1000 u/µl)
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EN0541, #EN0542 MSDS (English) MSDS (English-USA) MSDS (German)

Description
The RNase T1 is an endoribonuclease that specifically degrades single-stranded RNA at G residues. It cleaves the phosphodiester bond between 3'-guanylic residues and the 5'-OH residues of adjacent nucleotides with the formation of corresponding intermediate 2', 3'-cyclic phosphates (1). The reaction products are 3'-GMP and oligonucleotides with a terminal 3'-GMP.
The RNase T1 does not require metal ions for activity.

Source
E.coli cells with a cloned rntA gene of Aspergillus oryzae.

Molecular Weight
11.2 kDa monomer.

Applications

• Removal of RNA from DNA preparations.
• RNA sequencing (1).
• Ribonuclease protection assays. Used in conjunction with RNase A (2).
• Removal of RNA from recombinant protein preparations.
• Determination of the level of RNA transcripts synthesized in vitro from DNA templates containing a “G-less cassette” (3).

Quality Control
The absence of endodeoxyribonucleases, exodeoxyribonucleases and proteases confirmed by appropriate quality tests. Functionally tested for RNA digestion in the plasmid DNA purification procedure (in conjunction with RNase A).

Concentration
1000 u/µl

Definition of Activity Unit
One unit of the enzyme causes an increase in absorbance of 1.0 at 260 nm in 15 minutes when yeast RNA is hydrolyzed at 37°C and pH 7.5.
Enzyme activity is assayed in the following mixture: 50 mM Tris-HCl (pH 7.5), 2 mM EDTA, 3 mg/ml yeast RNA.

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

Inhibition and Inactivation

• Inhibitors: 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 (4).
• Inactivated by phenol/chloroform extraction.

Recommendations for Use

Reaction mixture for complete digestion of RNA: 50 mM Tris-HCl (pH 7.5), 1 mM EDTA and 0.2 u RNase T1/µg RNA. Incubate at 37°C for 30 minutes. RNase digestion mixture for RNase protection assay (2): 10 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM EDTA (pH 7.5), 40 µg/ml RNase A, 1000 u/ml RNase T1.

 

Related Products

RNase A, DNase and protease-free RNase A/T1 Mix GeneJET™ Plasmid Miniprep Kit Genomic DNA Purification Kit Proteinase K Water, nuclease-free

References

1. Takahashi K., Moore S., Ribonuclease T1, The Enzymes, V, (Boyer, P.D, ed.), Academic Press, New York, the third edition, vol. 15, 435-468, 1982.
2. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 7.63-7.74, 2001.
3. Sawadogo, M., Roeder, R.G., Factors involved in specific transcription by human RNA polymerase II: Analysis by a rapid and quantitative in vitro assay, Proc. Natl. Acad. Sci. USA, 82, 4394-4398, 1985.
4. Eun, H-M., Enzymology Primer for Recombinant DNA Technology, Academic Press, Inc., 1996.

Updated kovo 18, 2008 09:59