Description
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)
Storage temperature: –25°C to –15°C Molecular weight: 18.1 kDa Test Amount tested Specification Purity n/a >99% Specific activity n/a 625,000 U/mg Single-stranded exonuclease 500 U <5.0 % released Double-stranded exonuclease 500 U <1.0 % released Double-stranded endonuclease 500 U No conversion E. coli DNA contamination 500 U <10 copies Non-specific RNase 500 U No detectable non-specific RNase
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.
Usage Instructions 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. 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.
