BLIRT RNase H

RNase H hydrolyses specifically the phosphodiester bonds of RNA hybridized to DNA

RNase H
Name Part Number Pack Size
RNase H RT34-025 250 U (5 U/µl)
  RT34-125 1250 U (5 U/µl)

 

RNase H is a 18.9 kDa recombinant endoribonuclease purified from an Escherichia coli  strain, which over-expresses cloned RNase H gene (rnh). The enzyme hydrolyses specifically 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. 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 the removal of poly(A) tails on mRNAs after hybridization with oligo(dT) and also for the site-specific enzymatic cleavage of RNA.

Applications

- 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 hybridisation with oligo(dT)

- Site-specific cleavage of RNA

- Studies of in vitro polyadenylation reaction products

Storage buffer

20 mM Tris-HCl (pH 7.9), 100 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT, 50% (v/v) glycerol.

10x Reaction Buffer

200 mM Tris-HCl (pH 8.4), 500 mM KCl, 50 mM MgCl2, 200 mM DTT

Usage

- 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.

Additional information

- 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.

Quality control

RNase H is >90% pure as judged by SDS polyacrylamide gel. The absence of DNase activity has been confirmed using the relevant procedures. In addition, the functional quality is tested by RT and subsequent PCR and qPCR assays.

Unit definition

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.

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