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RNase H MBG (250 U)
Cat. No. / ID: RT34-025
5 U/μL concentration. Storage temperature: -20°C
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Quantity
250 U
1250 U
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The RNase H MBG is intended for molecular biology applications. This product is neither intended for the diagnosis, prevention or treatment of a disease, nor has it been validated for such use either alone or in combination with other products.
Need bulk, customized or optimized products for commercial purposes? We also offer support with logistics, compliance and more. Reach out to cooperate with QIAGEN Strategic Partnerships & OEM
Features
- Specifically hydrolyzes the phosphodiester bonds of RNA hybridized into DNA
- Prevents degradation of single and double-stranded DNA or un-hybridized RNA
- Improves sensitivity and prevents binding of amplification primers in a PCR reaction through binding of RNA to cDNA template
Product Details
RNase H is an 18.9 kDa recombinant endoribonuclease purified from an Escherichia coli strain, which over-expresses cloned RNase H gene (rnh).
It specifically hydrolyzes the phosphodiester bonds of RNA hybridized to DNA and produces 5´ phosphate-terminated oligoribonucleotides and single-stranded DNA. RNase H does not degrade single and double-stranded DNA or unhybridized RNA. It is a key enzyme in the removal of mRNA after first-strand cDNA synthesis.
It is supplied with 200 mM Tris-HCl (pH 8.4), 500 mM KCl, 50 mM MgCl2 and 200 mM DTT Reaction Buffer (10x).
One unit catalyzes the hydrolysis of 1 nmol of RNA in [3H]-labeled poly(A)×poly(dT) to acid-soluble ribonucleotides in a total reaction volume of 50 µL in 20 minutes at 37°C in 1x Reaction Buffer.
Performance
| Assay | Specification |
|---|---|
| Protein Purity | >90% |
| RNase contamination | None detected |
| DNase contamination | None detected |
| Proteases contamination | None detected |
Principle
Treating cDNA with RNase H prior to PCR can improve sensitivity as RNA bonded to the cDNA template may prevent binding of the amplification primers in a PCR reaction. RNase H treatment is often necessary when amplifying longer, full-length cDNA targets. In addition, RNase H is useful for removing poly(A) tails on mRNAs after hybridization with oligo(dT) and for the site-specific enzymatic cleavage of RNA.
Procedure
Quality Control
Protein purity is evaluated using Coomassie Blue detection assay by SDS-PAGE electrophoresis resulting in >90% purity. The relevant procedures have confirmed the absence of DNase, RNase and protease activity.
RNase and DNase contamination is evaluated by assessing RNase activity through gel electrophoresis following the incubation of RNA at 37°C.
Protease activity is also determined by SDS-PAGE electrophoresis.
Usage
Use 5 U of the enzyme to remove RNA from an RNA:DNA duplex after reverse transcription in a 20 μL reaction. If a 50 μL reaction is desired, 12.5 U of the enzyme is recommended.
The reaction mixture should be incubated at 37⁰C for 20 minutes.
Applications
This is used for applications such as:
- Removal of RNA after first strand cDNA synthesis (RT-PCR and qRT-PCR)
- Removal of mRNA prior to synthesis of second strand cDNA
- Removal of the poly(A) sequences of mRNA after hybridization with oligo(dT)
- Site-specific cleavage of RNA
- Studies of in vitro polyadenylation reaction products
Resources
Certificates of Analysis (1)