RNase H
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RNase H Highlights
RNase H is a non-specific endonuclease derived from E. coli. It cleaves RNA in an RNA:DNA hybrid by hydrolyzing the RNA phosphodiester bonds while leaving the DNA strand intact. RNase H does not hydrolyze single or double-stranded DNA or RNA.
- Efficient RNA cleavage of RNA:DNA substrates prepares cDNA for second strand synthesis
- Reduce material costs with a drop-in solution that delivers equivalent performance at a differentiated price-point
- Custom formats, including high concentration, support lyophilization applications
- Highly stringent enzyme manufacturing ensures quality performance across lots
Applications
- Cleavage of RNA for second-strand cDNA synthesis
- Applications that require RNA cleavage with an RNA:DNA substrate
- RT-PCR and qRT-PCR: removal of RNA after first strand cDNA synthesis
QC Specifications
| Description | Specification |
|---|---|
| Protein Purity Assay | ≥ 99% |
| Nonspecific RNase* | Not detectable |
| dsDNA Exonuclease Assay* | < 1% released |
| ssDNA Exonuclease Assay* | < 1% released |
| Phosphatase Contamination Assay* | < 1% released |
| DNA Contamination Assay (E. coli , mammalian, library)** | < 10 copies |
| Endonuclease Contamination Assay** | Not detectable |
| Unit Assay | Pass |
*As assessed using 600 U of enzyme input per assay.
**As assessed using 200 U of enzyme input per assay.
Properties
Unit definition: One unit is defined as the amount of RNase H that will hydrolyze 16 pmol of ribonucleotides from an RNA:DNA probe in 10 minutes at 30°C.
Reaction conditions:
50 mM Tris-HCl, pH 8.3
75 mM KCl
3 mM MgCl2
10 mM DTT
Storage buffer: 20 mM Tris-HCl, pH 7.4, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.01% Tween 20
Heat inactivation: 65°C for 20 min
Molecular weight: 17.6 kDa
BROCHURES
Precision Enzymes and Proteins Brochure
Custom Genomic Solutions Enzymes MDx DNA Polymerase I Equinox Library Amplification Equinox Uracil-Tolerant Library Amplification phi29 DNAP RNase Inhibitor RNase H StellarScript RT StellarScript HT RT StellarScript HT+ RT StellarTaq DNA Polymerase T4 DNA Ligase T4 DNAP T4 Polynucleotide Kinase T4 Gene 32 Protein Taq DNAPWatchmaker Genomics offers a portfolio of precision-engineered enzymes and proteins, designed to meet the demands of high-stringency applications. We provide tailored services such as custom fills, formats, packaging, and labeling to accommodate unique specifications.
PROTOCOLS
Watchmaker RNase H Technical Guide
Custom Genomic Solutions DNA RNA Enzymes RNase HProtocol for Watchmaker Genomics' RNase H (5 U/µL), which specifically degrades RNA by cleaving RNA in an RNA:DNA hybrid, hydrolyzing the RNA phosphodiester bonds while leaving the DNA strand intact.
| CONFIGURATION | |
|---|---|
| Kit contents | RNase H (5 U/µL) |
| Unit definition | One unit is defined as the amount of RNase H that will hydrolyze 16 pmol of ribonucleotides from an RNA:DNA probe in 10 minutes at 30°C. |
| Enzyme storage buffer | 20 mM Tris-HCl, pH 7.4, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.01% Tween 20 |
| QUALITY CONTROL | |
|---|---|
| Protein Purity Assay | ≥ 99% |
| Nonspecific RNase* | Not detectable |
| dsDNA exonuclease* | < 1% released |
| ssDNA exonuclease* | < 1% released |
| Phosphatase Contamination* | < 1% released |
| DNA Contamination** (E. coli, mammalian, library) | < 10 copies |
| Endonuclease** | Not detectable |
| Unit Assay* | Pass |
*As assessed using 600 U of enzyme input per assay.
**As assessed using 200 U of enzyme input per assay.
| SHIPPING AND HANDLING | |
|---|---|
| Shipping conditions | Ice packs |
| Storage | -20°C ± 5°C |
| Shelf life (off-the-shelf products) | ≥ 12 months |
| Shelf life (custom products) | ≥ 18 months |
What is RNase H?
RNase H is an endoribonuclease that degrades RNA by cleaving RNA in a RNA:DNA hybrid, hydrolyzing the RNA phosphodiester bonds while leaving the DNA strand intact.
On which side of the RNA nucleotide does RNase H cleave?
RNase H cleaves the 3'-O–P bond in the RNA backbone, resulting in a 3' hydroxyl group and a 5' phosphate on the cleavage products.
How is one unit of RNase H defined?
One unit is defined as the amount of RNase H that will hydrolyze 16 pmol of ribonucleotides from an RNA:DNA probe in 10 minutes at 30°C.
What is the origin of Watchmaker’s RNAse H?
RNase H is produced from a clone expressing the E. coli RNase H (rnh) gene.
What are the applications for the RNase H?
RNase H can be used in applications that require removal of RNA from an RNA:DNA substrate. An example includes removal of the RNA template after first strand cDNA synthesis.
What is the storage buffer for RNase H?
RNase H storage buffer comprises 20 mM Tris-HCl, pH 7.4, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 50% Glycerol, 0.01% Tween 20.
What reaction buffer does one use with RNase H?
RNase H is compatible with a standard 10X reaction buffer: 500 mM Tris-HCl, pH 8.3, 750 mM KCl, 30 mM MgCl2, 100 mM DTT. This buffer is not supplied in the kit.
What is the optimal temperature for RNase H activity?
The optimal temperature for RNase H activity is 37°C.
Is RNase H treatment required before PCR amplification?
For most RT-PCR and RT-qPCR reactions, RNase H treatment is not required.
What are the recommended reaction setup conditions?
Please reference our technical guide for instructions for use.
1. On ice, combine components as specified:
| Component | Final Concentration | Volume (50 µL) |
|---|---|---|
| 10X reaction buffer | 1X | 5 µL |
| RNA:DNA duplex | 1 - 2000 ng | variable |
| RNase H (5 U/µL) | 0.05 U/µL | 0.5 µL |
| Nuclease-free H2O | Up to 50 µL |
2. Incubate as follows:
| Purpose | Temperature | Time |
|---|---|---|
| RNA cleavage | 37°C | 20 minutes |
| Inactivation | 65°C | 20 minutes |
Can RNase Inhibitor be inactivated?
RNase H can be inactivated by heating at 65°C for 20 minutes.
What is the extinction coefficient of RNase H?
The extinction coefficient for RNase H is 2.290 mL/mg*cm. If measuring protein concentration (A280), we recommend you use the protein’s extinction coefficient to ensure an accurate reading.
Do you offer custom formats?
Yes! Watchmaker offers customer fills, packaging, concentrations, and labeling - including private label - designed to meet your unique needs. We offer flexible terms to serve organizations of any size, and our right-sized processes enable rapid turnaround time on customization. Please contact sales@watchmakergenomics.com to learn more about our capabilities.
Are you ISO 13485-certified?
Yes! Our Quality Management System has achieved ISO 13485:2016 Certification. Our certificate has been awarded for the design, development, manufacture, contract manufacture, and support of high performing reagents for genomics applications in medical research. Download the certificate here.
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| Description | 0.5 kU | |
|---|---|---|
| RNase H (5 U/µL) | 7K0131-100UL | Request a quote |
Please contact sales@watchmakergenomics.com to inquire about custom kit configurations.
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