RNase T1 is an endoribonuclease derived from Aspergillus oryzae. It specifically cleaves single-stranded RNA after guanosine residues (G), generating 3'-phosphate termini. RNase T1 forms nucleoside 2',3'-cyclic phosphate intermediates, cleaving the phosphodiester bond between the 3'-guanosine residue and the adjacent nucleoside 5'-OH group to produce oligonucleotides terminating in 3'-GMP and 3'-GMP.
The activity of RNase T1 is independent of metal ions.
Components List
R1522664
| Components | Appearance | 2KU | 5×2KU | Storage | | R1522664A | RNase T1 (500 U/μl) | Liquid | 200ul | 5×200µl | -20℃. Avoid freeze/thaw cycle. | | R1522664B | 10× RNase T1 Buffer | Liquid | 1ml | 5×1ml | -20℃. Avoid freeze/thaw cycle. |
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Unit Definition
One unit (U) is defined as the amount of enzyme required to produce an absorbance change of 1.0 at A260 using yeast RNA as the substrate at 37 °C and pH 7.5.
Applications
- Removal of RNA contaminant from DNA preparations.
- Removal of RNA contaminant from recombinant protein preparations.
- RNA sequencing.
- Use in ribonuclease protection assays (in combination with RNase A).
- Quantitation of transcription levels from DNA templates with no or low guanine content.
Quality Control
Protein Purity
SDS-PAGE analysis confirms protein purity of no less than 95%.
Non-specific Endonuclease Activity
500 U of RNase T1 was incubated with 200 ng supercoiled plasmid DNA in a 20 μL reaction system at 37 °C for 4 hours. Agarose gel electrophoresis showed less than 20% of the plasmid DNA was converted to nicked or linear form.
DNase Activity
500 U of RNase T1 was incubated with 15 ng double-stranded DNA fragments in a 20 μL reaction system at 37 °C for 16 hours. Agarose gel electrophoresis showed no degradation of the dsDNA fragments.
Usage Protocol
1. Reaction system
| Component | Volume | Final Concentration |
|---|
| ssRNA | 1~10 μg | 50~500 ng/μL | | 10× RNase T1 Buffer | 2 μL | 1× | | RNase Inhibitor (40 U/μL)* | 1 μL | 2 U/μL | | RNase T1 (500 U/μL) | 0.4 μL | 10 U/μL | | Nuclease-Free Water | up to 20 μL | - |
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Note: To prevent degradation of the substrate RNA caused by contamination with environmental RNases such as RNase A, it is recommended to add RNase Inhibitor (Cat.No.: R350859) to the reaction system. The substrate RNA should be added only after the RNase Inhibitor has been added and thoroughly mixed.
2. Recommended reaction conditions: Incubate at 37 °C for 15 minutes. The incubation time can be appropriately extended or shortened according to actual conditions to achieve optimal digestion results.
Notes
- The activity of RNase T1 can be inhibited by various metal ions, including Mg²⁺ (100 mM MgCl₂ inhibits approximately 40% of activity), Ca²⁺ (10 mM CaCl₂ inhibits approximately 30% of activity), Zn²⁺, Fe²⁺, Cu²⁺, etc. In addition, Guanylyl 2′-5′ guanosine is a specific inhibitor of RNase T1.
- Heat inactivation of RNase T1 is reversible. It is recommended to remove RNase T1 by column purification or phenol/chloroform extraction.
- RNase T1 is stable when heated at 100℃ for 10 min in solutions at pH 6.0, but unstable in alkaline solutions at pH > 9.0.
- For your safety and health, please wear a lab coat, disposable gloves, and a face mask during the experiment.
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