Thermostable RNase HII

This product is derived from the extreme thermophilic archaeon Pyrococcus abyssi (P. abyssi) and obtained after recombinant expression and purification.Thermostable RNase HII is an endoribonuclease that recognizes and cleaves the RNA strand in RNA/DNA hybrids, but does not cleave ssDNA or dsDNA, and has very low activity against single-stranded RNA.Thermostable RNase HII cleaves at the 5' side of single ribonucleotide residues, generating a 5'-phosphate group and a 3'-hydroxyl group after cleavage.This Thermostable RNase HII exhibits optimal activity at 60–80 °C, retains activity between 37–95 °C, and shows low activity at room temperature.It is extremely thermostable: after incubation at 95 °C for 15 minutes, there is almost no loss of activity, making it fully compatible with various PCR reaction systems.Components ListT1522666ComponentsAppearance200UStorageT1522666AThermostable RNase HII (2 U/μl)Liquid100μl-20℃. Avoid freeze/thaw cycle.T1522666BHII Dilution BufferLiquid2×1ml-20℃. Avoid freeze/thaw cycle.T1522666C10× RHII BufferLiquid1ml-20℃. Avoid freeze/thaw cycle.Unit DefinitionOne unit (U) is defined as the amount of enzyme required to cleave 100 pmol of chimeric DNA/RNA hybrid duplex containing a single ribonucleotide in 30 minutes at 60 °C.Recommended Reaction Conditions1× RHII Buffer; incubation at 60 °C.Inactivation Conditions1% (final concentration) SDS, incubation at 95 °C for 15 minutes.ApplicationsRNase HII-dependent PCR (rhPCR).Reducing or eliminating primer-dimer formation in PCR reactions.Improving the accuracy of multiplex PCR products.Distinguishing paralogous homologous genes.SNP detection and rare allele detection.Degrading the RNA portion of Okazaki fragments.LAMP high-sensitivity probe-based detection.Generating double-strand breaks at incorporated ribonucleotide sites when used with T7 Endonuclease I.Quality ControlProtein PuritySDS-PAGE analysis confirms protein purity of no less than 95%.Non-specific Endonuclease Activity2 U of Thermostable RNase HII 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 Activity2 U of Thermostable RNase HII 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.RNase Activity2 U of Thermostable RNase HII was incubated with 500 ng RNA in a 10 μL reaction system at 37 °C for 1 hour. Agarose gel electrophoresis showed more than 90% of the RNA remained intact.Host DNA ResidueThe residual host DNA level in this product is less than 1 copy per 2 U, as determined by quantitative PCR (Method III) according to the Chinese Pharmacopoeia 2020 Edition Part IV General Rule 3407.Usage ProtocolExample protocol for rhPCR:(1) Reaction system using the company's Taq-Plus PCR Master Mix (2×) (Cat. No.: T295072):ReagentVolumeForward Primer (10 μM)1 μlReverse Primer (10 μM)1 μlTemplate DNAx μlTaq-Plus PCR Master Mix (2×)25 μlThermostable RNase HII20~200 mUddH₂OUp to 50 μl（2）Mix gently by pipetting up and down or flicking the tube (do not vortex), then briefly centrifuge to collect all liquid droplets on the tube wall.（3）Set up the PCR reaction. 2. rhPCR Primer DesignPrecautionsWhen using this enzyme for rhPCR, PCR primers with a 3'-blocking group are required.This enzyme is active at 37–80 °C, and its dosage can be adjusted according to different applications.This enzyme is compatible with most PCR systems and has no strict requirements on Mg²⁺ concentration; it can function normally within the range of 1–6 mM.

Specifications & Purity: This product is derived from the extreme thermophilic archaeon Pyrococcus abyssi (P. abyssi) and obtained after recombinant expression and purification.Thermostable RNase HII is an endoribonuclease that recognizes and cleaves the RNA strand in RNA/DNA hybrids, but does not cleave ssDNA or dsDNA, and has very low activity against single-stranded RNA.Thermostable RNase HII cleaves at the 5' side of single ribonucleotide residues, generating a 5'-phosphate group and a 3'-hydroxyl group after cleavage.This Thermostable RNase HII exhibits optimal activity at 60–80 °C, retains activity between 37–95 °C, and shows low activity at room temperature.It is extremely thermostable: after incubation at 95 °C for 15 minutes, there is almost no loss of activity, making it fully compatible with various PCR reaction systems.Components ListT1522666ComponentsAppearance200UStorageT1522666AThermostable RNase HII (2 U/μl)Liquid100μl-20℃. Avoid freeze/thaw cycle.T1522666BHII Dilution BufferLiquid2×1ml-20℃. Avoid freeze/thaw cycle.T1522666C10× RHII BufferLiquid1ml-20℃. Avoid freeze/thaw cycle.Unit DefinitionOne unit (U) is defined as the amount of enzyme required to cleave 100 pmol of chimeric DNA/RNA hybrid duplex containing a single ribonucleotide in 30 minutes at 60 °C.Recommended Reaction Conditions1× RHII Buffer; incubation at 60 °C.Inactivation Conditions1% (final concentration) SDS, incubation at 95 °C for 15 minutes.ApplicationsRNase HII-dependent PCR (rhPCR).Reducing or eliminating primer-dimer formation in PCR reactions.Improving the accuracy of multiplex PCR products.Distinguishing paralogous homologous genes.SNP detection and rare allele detection.Degrading the RNA portion of Okazaki fragments.LAMP high-sensitivity probe-based detection.Generating double-strand breaks at incorporated ribonucleotide sites when used with T7 Endonuclease I.Quality ControlProtein PuritySDS-PAGE analysis confirms protein purity of no less than 95%.Non-specific Endonuclease Activity2 U of Thermostable RNase HII 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 Activity2 U of Thermostable RNase HII 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.RNase Activity2 U of Thermostable RNase HII was incubated with 500 ng RNA in a 10 μL reaction system at 37 °C for 1 hour. Agarose gel electrophoresis showed more than 90% of the RNA remained intact.Host DNA ResidueThe residual host DNA level in this product is less than 1 copy per 2 U, as determined by quantitative PCR (Method III) according to the Chinese Pharmacopoeia 2020 Edition Part IV General Rule 3407.Usage ProtocolExample protocol for rhPCR:(1) Reaction system using the company's Taq-Plus PCR Master Mix (2×) (Cat. No.: T295072):ReagentVolumeForward Primer (10 μM)1 μlReverse Primer (10 μM)1 μlTemplate DNAx μlTaq-Plus PCR Master Mix (2×)25 μlThermostable RNase HII20~200 mUddH₂OUp to 50 μl（2）Mix gently by pipetting up and down or flicking the tube (do not vortex), then briefly centrifuge to collect all liquid droplets on the tube wall.（3）Set up the PCR reaction. 2. rhPCR Primer DesignPrecautionsWhen using this enzyme for rhPCR, PCR primers with a 3'-blocking group are required.This enzyme is active at 37–80 °C, and its dosage can be adjusted according to different applications.This enzyme is compatible with most PCR systems and has no strict requirements on Mg²⁺ concentration; it can function normally within the range of 1–6 mM.

Grade: Recombinant;Suitable for molecular biology;EnzymoPure™

EC Number: 998-948-1

PubChem CID: 139291116

Storage Temperature: Store at -20°C;Avoid repeated freezing and thawing

Shipped in: Ice chest + Ice pads

UPC:

12352204

Weight:

7.06 Ounces

HazmatClass:

Yes

Quantity:

1

Unit of Measurement:

EA

WeightUOM:

LB

MPN:

T1522666-200U

Country of Origin:

China

CAS:

9050-76-4

Product Size:

200U

Harmonized Tariff Code:

2931.90.90