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 catalyses 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
200.0 µg/ml BSA
Storage Buffer Conditions of RNaseH in Trehalose. Same as above, except for 1.0 M Trehalose instead of Glycerol.
Quality Control
1. 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.
2. 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.
3. Specific Activity:
The specific activity of the E. coli RNase H is no less than 300,000 units per mg.
References:
1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd Ed.), 8.64 – 8.65
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 catalyses 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
200.0 µg/ml BSA
Storage Buffer Conditions of RNaseH in Trehalose. Same as above, except for 1.0 M Trehalose instead of Glycerol.
Quality Control
1. 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.
2. 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.
3. Specific Activity:
The specific activity of the E. coli RNase H is no less than 300,000 units per mg.
References:
1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd Ed.), 8.64 – 8.65
