RNase H

RNase H (rnh) is an endoribonuclease that degrades the RNA strand of RNA:DNA hybrid molecules. RNase H digestion produces ribonucleotide molecules with 5-phosphate and 3-hydroxyl termini. RNase H is nearly inactive against single- and double-stranded RNA molecules.

RNase is supplied in 20 mM Tris-HCl, 100 mM KCl, 0.1mM DTT, 10 mM MgCl2, 0.1 mM EDTA and 50% glycerol; pH 7.9 at 25⁰C.

The 10X RNase H Buffer (cat. no. B9220) contains 500 mM Tris-HCl, 750 mM KCl, 30 mM MgCl₂ and 100mM DTT; pH 8.3 at 25⁰C.

OEM by QIAGEN offers bulk manufacturing of RNase H in custom formulations.

Polymerase properties

Storage temperature: –25°C to –15°C

The source of the protein is 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 an 3H-labeled DNA:RNA hybrid molecule into acid-soluble material in 20 minutes at 37°C.

The molecular weight of the protein is 18,053 Daltons.

Quality control analysis

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

Protein concentration (OD₂₈₀) is determined by OD₂₈₀ 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 plasmid DNA and 10 µl enzyme solution that was 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.

• RT-PCR
• cDNA synthesis
• RT-qPCR