rDNase I, RNase-Free

Source: Recombinant

Applications:

1. Removal of DNA from protein and RNA preparations.
2. Degradation of template DNA from RNA after in vitro transcription.
3. Removal of genomic DNA prior to RT-PCR.
4. Nick translation with DNA Polymerase I (PN 70010).

Description:
Recombinant DNase I, RNase-Free, is overexpressed and purified from a non-animal host, Pichia pastoris, which is vastly lower in endogenous RNase levels than pancreatic tissue, the source for bovine DNase I. Hence, the recombinant enzyme is readily purified without any detectable RNase contamination.

DNase I is an endonuclease that hydrolyzes phosphodiester linkages in DNA to yield di-, tri-, and oligonucleotides with a 5’-phosphate and a 3’-hydroxyl termini⁽¹⁾.  DNase I can cleave dsDNA, ssDNA, chromatin and DNA:RNA hybrids. However, the cleavage rates for ssDNA and DNA:RNA hybrids are much lower than dsDNA. This enzyme is totally inactive against RNA⁽²⁾. For maximum activity, DNase I requires both Ca²⁺ and Mg^{2+ (3)}. Ca²⁺ stabilizes DNase I to maintain the active conformation and Mg²⁺ is required for activity.

Properties:
Molecular Weight: ~30,000 Da
Activators: Mg²⁺ and Ca²⁺ for maximum activity
Inhibitors: EDTA and EGTA
Heat Inactivation: 75°C for 10 min at a 0.1 unit/μl DNase I concentration. Add EDTA to a final concentration of 5mM before heating to avoid chemical scission of RNA.

Purity:
Tested for contaminating ribonucleases and proteases.

Storage Buffer:
20mM Tris-HCl, pH 7.6, 50mM NaCl, 2mM CaCl₂, 2mM MgCl₂, 1mM DTT, 0.1 mg/ml pefabloc SC, and 50% glycerol.

Assay Conditions:
100 µg calf thymus DNA is incubated in 1X reaction buffer with 40 to 70 units of rDNase I at 25°C. The absorbance increase is measured at 260 nm. 

Unit Definition:
One unit is the amount of enzyme that causes an absorbance increase of 0.001 per min in 1 ml at 260nm at 25°C.

Concentration:
10 units/μl

10X rDNase I Reaction Buffer (1 ml included, PN 78416):
400mM Tris-HCl, pH 7.9, 100mM NaCl, 60mM MgCl₂, 10mM CaCl₂.

Stop Solution (1 ml included, PN 78317):
50mM EDTA, pH 8.0.

References:

1. KUNITZ, M. (1950) J. Gen. Physiol. 33, 349.
2. SUTTON, D. H., CONN, G. L. , BROWN, T., AND LANE, A. N. (1997) Biochem. J. 321, 481-486.
3. CLARK, R. AND EICHHORN, G. L. (1974) Biochem. 13, 5098.

Storage:
Shipped on dry ice. Store at -20°C.