RNase H

- Thermal inactivation at 65°C in 10 min
- LO certified

Catalog#	Size, concentration	Supplied with:	Certificate of Analysis	MSDS
Catalog#	Size, concentration	10X Reaction Buffer	Certificate of Analysis	MSDS
EN0201	100 u (5 u/µl)	1.00 ml	EN0201
EN0202	500 u (5 u/µl)	1.00 ml	EN0202

Product information

Applications

Removal of mRNA prior to synthesis of second strand cDNA (1).
RT-PCR and qRT-PCR: removal of RNA after first strand cDNA synthesis.
Removal of the poly(A) sequences of mRNA after hybridization with oligo(dT) (2).
Site-specific cleavage of RNA (3).
Studies of in vitro polyadenylation reaction products (4).

Description

Ribonuclease H (RNase H) specifically degrades the RNA strand in RNA-DNA hybrids, see Fig.1. It does not hydrolyze the phosphodiester bonds within single-stranded and double-stranded DNA and RNA.

Source

E.coli MRE-600 cells.

Molecular Weight

18.4 kDa monomer.

Definition of Activity Unit

One unit of the enzyme catalyzes the formation of 1 nmol of acid soluble products in 20 min at 37°C.

Enzyme activity is assayed in the following mixture: 20 mM Tris-HCl (pH 7.8), 40 mM KCl, 8 mM MgCl₂, 1 mM DTT, 24 µM [³H]-poly(A)·poly(dT), 0.03 mg/ml BSA, 4% (v/v) glycerol.

Storage Buffer

The enzyme is supplied in:
25 mM HEPES-KOH (pH 8.0), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA and 50% (v/v) glycerol.

10X Reaction Buffer

200 mM Tris-HCl (pH 7.8), 400 mM KCl, 80 mM MgCl₂, 10 mM DTT.

Quality Control

The absence of endo-, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests.

Inhibition and Inactivation

Inhibitors: metal chelators, SH-blocking reagents.
Inactivated by heating at 65°C for 10 min.

Figure 1. RNase H activity.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

II. Second Strand cDNA synthesis

Perform first strand cDNA synthesis reaction according to recommendations provided for a specific reverse transcriptase.

Add the following (on ice) to 20 µl of first strand cDNA synthesis reaction mixture:

10X reaction buffer for DNA Polymerase I 8 µl

RNase H 0.2 µl (1 u)

DNA Polymerase I 3 µl (30 u)

Water, nuclease-free 88.8 µl

Total volume 100 µl
Gently vortex and briefly centrifuge.
Incubate at 15°C for 2 hours. Do not let the temperature rise above 15°C.
Add 2.5 µl (12.5 u) of T4 DNA Polymerase and incubate at 15°C for 5 min.
Terminate the reaction by adding 5 µl of 0.5 M EDTA, pH 8.0.

Phenol/chloroform purified blunt-end cDNA can be used for further cloning related procedures e.g. adapter ligation, phosphorylation, size fractionation, ligation and transformation.

References

Gubler, U., Hoffman, B.J., A simple and very efficient method for generating cDNA libraries, Gene, 25, 263-269, 1983.
Davis, R., et al., Tandemly repeated exons encode 81-base repeats in multiple, developmentally regulated Schistosoma mansoni transcripts, Mol. Cell Biol., 8, 4745-4755, 1988.
Donis-Keller, H., Site specific enzymatic cleavage of RNA, Nucleic Acids Res., 7, 179-192, 1979.
Goodwin, E.C., Rottman, F.M., The use of RNase H and poly(A) junction oligonucleotides in the analysis of in vitro polyadenylation reaction products, Nucleic Acids Res., 20, 916, 1992.

Related products