Qiagen, Y9220L, RNase H (5000 U)

RNase H (1 mL at 5000 U/mL) and 10X RNase Buffer (2 x 1.5 mL)

Features

- Useful for removing mRNA during second-strand cDNA synthesis
- Produces ribonucleotide molecules with 5’-phosphate and 3’-hydroxyl termini

Product Details

E. coli RNase H (rnh) is an endoribonuclease that degrades the RNA strand of RNA/DNA hybrid molecules (1,2). RNase H digestion produces ribonucleotide molecules with 5’-phosphate and 3’-hydroxyl termini. RNase H is nearly inactive against single or double-stranded RNA molecules.

Supplied in: 20 mM Tris-HCl, 100 mM KCl, 0.1 mM DTT, 10 mM MgCl 2 , 0.1 mM EDTA, 50% glycerol (pH 7.9 at 25°C)

Supplied with: 10X RNase H Buffer (B9220): 500 mM Tris-HCl, 750 mM KCl, 30 mM MgCl 2 , 100 mM DTT (pH 8.3 at 25°C)

Performance

Test: Amount tested
Purity: n/a
Specific activity: n/a
Single-stranded exonuclease: 500 U
Double-stranded exonuclease: 500 U
Double-stranded endonuclease: 500 U
E. coli DNA contamination: 500 U
Non-specific RNase: 500 U

- Storage temperature: –25°C to –15°C
- Molecular weight: 18.1 kDa

Principle

The enzyme is purified from a recombinant E. coli strain carrying the RNase H (rnh) gene from E. coli .

One unit is defined as the amount of enzyme that will hydrolyze 1 nmol of RNA from a 3 H-labeled DNA:RNA hybrid molecule into acid-soluble material in 20 minutes at 37°C.

Procedure

Components: Final Concentration
Nuclease-free water: N/A
10X RNase H Buffer (B9220): 1X
RNA: DNA duplex: 2 µg
RNase H (Y9220L): 5 U
Total Volume =: 100 µL

- Set up the following reaction mixture in a total volume of 100 µL: Components Final Concentration Volume Nuclease-free water N/A X µL 10X RNase H Buffer (B9220) 1X 10 µL RNA: DNA duplex 2 µg X µL RNase H (Y9220L) 5 U 1 µL Total Volume = 100 µL
- Incubate the reaction at 37°C for 20 minutes.
- Stop the reaction by adding 1 µL of 0.5 M EDTA.

Usage Instructions

Quality Control

Unit activity is measured using a 2-fold serial dilution method. Dilutions of the enzyme were made in 1X RNase H reaction buffer and added to 50 µL reactions containing 3 H-labeled poly(rA), poly (dT) DNA, and 1X RNase H Buffer. Reactions were incubated for 20 minutes at 37°C, plunged on ice, and release of TCA soluble counts was analyzed.

Protein concentration (OD 280 ) is determined by OD 280 absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the protein of interest band in the diluted sample.

Single-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C.

E. coli 16S rDNA contamination is evaluated using 5 µL replicate samples of enzyme solution denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

Non-specific RNase contamination is assessed using the RNase Alert kit (Integrated DNA Technologies), following the manufacturer’s guidelines.

Applications

- RT-PCR
- cDNA synthesis
- RT-qPCR

This product is available for molecular biology applications such as:

References

1. Donnis-Keller, H. (1979) Nucl. Acids Res., 7, 179. 2. Schultz, S.J. and Champoux, J.J. (2008) Virus Res., 134(1-2): 86-103.