Unit Definition:
One unit is the amount of enzyme required to completely degrade 1 μg of plasmid DNA in 10 min at 37°C.
One functional Dnase I unit is approximately equivalent to 0.3 Kunitz unit.
Applications:
· Preparation of DNA-free RNA (degradation of contaminating DNA after RNA isolation).· Preparation of DNA–free RNA prior RT-PCR and RT-qPCR.· Removal of template DNA following in vitro transcription.
· Studies of DNA-protein interactions (footprinting).
· DNA labeling by nick-translation.
· Production of random fragments (generation of libraries).
Enzyme activity: DNase I requires Ca2+ and Mg2+ for hydrolyzing double-stranded DNA. In the presence of Mg2+, DNase I cleaves each strand of double-stranded DNA independently in a statistically random fashion (recommended Reaction Buffer I). In the presence of Mn2+, the enzyme cleaves both DNA strands at approximately the same site, producing DNA fragments with blunt-ends or with overhang termini of only one or two nucleotide (recommended Reaction Buffer II).
10 x Reaction Buffer I:
100 mM Tris-HCl, 25 mM MgCl2, 100 mM CaCl2, pH 7.4 @ 25°C.
10 x Reaction Buffer II:
100 mM Tris-HCl, 100 mM CaCl2, pH 7.4 @ 25°C. Supplemented with
100 mM MnCl2.
Inactivation: Inactivated by heating at 65°C for 10 min in the presence of EDTA or EGTA.
Inhibitors: metal chelators (EGTA, EDTA), transition metals, SDS, reducing agents (DTT, β-mercaptoethanol).
References:
1. Kunitz, M (1950) J. Gen Physiol 33: 349-362.
2. Vanecko, S and Laskowski, M (1961). J Biol Chem 236: 3312-3316.
3. Although it is not required for most applications please see the additional DNase I digestion conditions in Manual for GeneMATRIX
UNIVERSAL DNA/RNA/Protein Purification Kit (E3597) and GeneMATRIX UNIVERSAL RNA Purification Kit (E3598).
4. Sanyal, A., et al., An effective method of completely removing contaminating genomic DNA from an RNA sample to be used for PCR. Mol. Biotechnol., 8, 135-137, (1997).
5. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
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