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RNAi and RNA Enzymes >
RNases >
RNase H |
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Prices are in US dollars and valid only for US orders.
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- Isolated from a recombinant source
- Supplied with 10X Reaction Buffer
Description:
RNase H (Ribonuclease H ) is an endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA which is hybridized to DNA. This enzyme does not digest single or double-stranded DNA.
Source:
An E. coli strain that carries the cloned RNase H gene (rnh) from Escherichia coli
Applications:- Removal of poly(A) tails of mRNA hybridized to poly(dT)
- Removal of mRNA during second strand cDNA synthesis
Reagents Supplied:
RNase H Reaction Buffer (10X)
Enzyme Properties
Heat Inactivation:
65°C for 20 minutes
Reaction & Storage Conditions
Reaction Conditions:
1X RNase H Reaction Buffer
Incubate at
37°C.
1X RNase H Reaction Buffer:
50 mM Tris-HCl
75 mM KCl
3 mM MgCl2
10 mM Dithiothreitol
pH 8.3 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme that will hydrolyze 1 nmol of the RNA in [3H]-labeled poly(rA).poly(dT), to acid-soluble ribonucleotides in a total reaction volume of 50 µl in 20 minutes at 37°C in 1X RNase H Reaction Buffer with 10 nmol [3H]-labeled poly(rA) and 12.5 µg poly(dT).
Concentration:
5,000 units/ml
Storage Conditions:
20 mM Tris-HCl
100 mM KCl
10 mM MgCl2
0.1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Purified free of contaminating endonucleases and exonucleases.
SS DNA Exonuclease Activity:
Incubation of 50 units of enzyme with 1 μg sonicated and denatured
[3H]-DNA (105 cpm/μg) for 30 minutes at 37ºC
in 50 μl reaction buffer released < 0.1% radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 50 units of RNase H with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
RNase Assay:
Incubation of a 50 μl reaction containing 50 units of RNase H with 1
μg of [3H]-labeled poly(rA) for 1 hour at 37ºC
resulted in no detectable degradation of the RNA as determined by agarose gel electrophoresis.
References
- Gubbler, U. and Hoffman, B.J. (1983) Gene, 25, 263-269.
- Davis, R. et al. (1988) Cell Biol., 8, 4745-4755.
- Donnis-Keller, H. (1979) Nucl. Acids Res., 7, 179.
- Goodwin, E.C. and Rottman, F.M. (1992) Nucl. Acids Res., 20, 916.
Reagents Sold Separately
RNase H Reaction Buffer
Companion Products
ProtoScript® First Strand cDNA Synthesis Kit
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