Description
Ribonuclease H from E.coli
Ribonuclease H is an endonuclease which specifically degrades the RNA moiety of a DNA:RNA hybrid, without affecting the unhybridized RNA. It does not affect double-stranded RNA or DNA. It is provided in a 50% glycerol storage buffer. It is also available in a Trehalose Buffer (Suitable for lyophilization). Concentration 1000 U/mL.
Please inquire for custom concentrations and bulk quantities.
Applications:
Enabling the synthesis of second-strand cDNA by removal of the RNA
Used in conjunction with AMV RT and T7 RNA Polymerase in amplification of RNA
Unit Definition:
One unit of RNaseH is that amount of enzyme which catalysis the production of one nmol of acid-soluble nucleotide in 20 minutes at 37ºC using the following reaction conditions:
40 mM TrisHCl, pH 7.5
1.0 µM
[3H]-poly (rA):24 µM poly(dT)
4.0 mM MgCl2
1.0 mM DTT
30 µg/mL BSA
4.0% glycerol
Storage Buffer Conditions of RNaseH in Glycerol:
20:0 mM TrisHCl, pH 7.5
300 mM KCl
20.0 mM Magnesium Acetate
7.0 mM EDTA
1.0 mM Dithiothreitol
50% Glycerol
0.2% Triton X100
Storage Buffer Conditions of RNaseH in Trehalose. Same as above, except for 1.0 M Trehalose instead of Glycerol.
Quality Control:
DNase Activity:
One-half µg of Hae fragments of Phi X-174 DNA is incubated at 37ºC with 2.5 units of RNase H for 3 hours, and then electrophoresed in a native agarose gel simultaneously with control positive DNase 1 reactions. No more than the equivalent of 2.5 X 10 E-4 unit of DNase 1 is detected.
Ribonuclease Activity:
One microgram of an RNA Ladder is incubated for 2 hours at 37ºC with 4.0 units of RNase H, and then electrophoresed in a native agarose gel simultaneously with control positive RNase 1A reactions. No more than the equivalent of 8 X 10-8 unit of RNase 1A is detected.
Specific Activity:
The specific activity of the E. coli RNase H is no less than 300,000 units per mg.
References:
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd Ed.), 8.64 – 8.65
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