RNase H is a 18.9 kDa recombinant endoribonuclease purified from an Escherichia coli strain. The enzyme hydrolyses specifically the phosphodiester bonds of RNA hybridized to DNA and produces 5´ phosphate-terminated oligoribonucleotides and single-stranded DNA. The enzymes 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. 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 treatment is often necessary when amplifying longer, full-length cDNA targets. In addition, this enzymes is useful for the removal of poly(A) tails on mRNAs after hybridization with oligo(dT) and also for the site-specific enzymatic cleavage of RNA.
- Recombinant endoribonuclease over-expressed in E. coli.
- Hydrolyses specifically the phosphodiester bonds of RNA hybridized to DNA.
- Does not degrade single and double-stranded DNA or un-hybridized RNA.
- 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.
- 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.
One unit catalyses 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 min at 37°C in 1x Reaction Buffer.
RNase H (RT34) - Manual
Use 5 U of enzyme to remove RNA from a RNA:DNA duplex after reverse transcription in a 20 μl reaction. If 50 μl reaction is desired, the use of 12.5 U of enzyme is recommended.
The reaction mixture should be incubated at 37⁰C for 20 minutes.
Store at -20°C.
Shipping on dry or blue ice.
10x Reaction Buffer
200 mM Tris-HCl (pH 8.4), 500 mM KCl, 50 mM MgCl2, 200 mM DTT
- The activity of RNase H is inhibited by metal chelators (e.g. EDTA) and sulfhydryl SH-blocking reagents.
- Inactivate enzyme by heating at 65°C for 10 min.
RNase H is >90% pure as judged by SDS polyacrylamide gel. The absence of DNase activity has been confirmed using the relevant procedures.
RNase H (RT34) - MSDS
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