Home | Molecular Biology | Molecular Biology Applications | In vitro Transcription | In vitro Transcription Enzymes

DNase I, RNase-free

recomb.gif hc.gif yes65_10_edta.gif

An endonuclease that nonspecifically cleaves single- and double-stranded DNA to release di-, tri-, and oligonucleotides with 5'-phosphate and 3'-OH groups.
  
Loading...

Thermo Scientific DNase I, RNase-free is an endonuclease that digests single- and double-stranded DNA. It hydrolyzes phosphodiester bonds producing mono- and oligodeoxyribonucleotides with 5'-phosphate and 3'-OH groups.

The enzyme activity is strictly dependent on Ca2+ and is activated by Mg2+ or Mn2+ ions.

  • In the presence of Mg2+, DNase I cleaves each strand of dsDNA independently in a statistically random fashion (see Reference 1).
  • 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 nucleotides (see Reference 1).

Highlights

  • Recombinant enzyme
  • Purified from non-animal host with a lower level of intrinsic RNases

Applications

  • Preparation of DNA-free RNA (see Reference 1)
  • Removal of template DNA following in vitro transcription (see Reference 1)
  • Preparation of DNA-free RNA prior to RT-PCR and RT-qPCR (see Reference 2)
  • DNA labeling by nick-translation in conjunction with DNA Polymerase I (see Reference 1)
  • Studies of DNA-protein interactions by DNase I, RNase-free footprinting (see Reference 1)
  • Generation of a library of randomly overlapping DNA inserts. Reaction buffer containing Mn2+ is used (see Reference 3)

Note

DNase I is sensitive to physical denaturation. Mix gently by inverting the tube. Do not vortex.

  
Storage Condition-20 C
HazardousNo
10X Reaction Buffer with MgCl2100 mM Tris-HCl (pH 7.5 at 25°C), 25 mM MgCl2, 1 mM CaCl2.
10X Reaction Buffer without MnCl2100 mM Tris-HCl (pH 7.5 at 25°C), 1 mM CaCl2. Recommended concentration of MnCl2 in 1X reaction buffer is 10 mM.
Definition of Activity Unit
  • One unit of the enzyme completely degrades 1 µg of plasmid DNA in 10 min at 37°C.
  • Enzyme activity is assayed in the following mixture: 40 mM Tris-HCl (pH 8.0), 10 mM MgSO4, 1 mM CaCl2, 1 µg of pUC19 DNA.
  • One DNase I unit is approximately equivalent to 0.3 Kunitz unit (see Reference 4).
InactivationInactivated by heating at 65°C for 10 min in the presence of EGTA or EDTA (use at least 1 mol of EGTA/EDTA per 1 mol of Mn2+/Mg2+ (5)).
InhibitionInhibitors: metal chelators, transition metals (e.g., Zn) in millimolar concentrations, SDS (even at concentrations less than 0.1%), reducing agents (DTT and beta-mercaptoethanol), ionic strength above 50 to 100 mM.
Molecular Weight29 kDa monomer
Quality Control
  • The absence of ribonucleases confirmed by appropriate quality test.
  • Functionally tested for digestion of template DNA after in vitro transcription.
SourceE. coli cells with a cloned gene encoding bovine DNase I.
Storage BufferThe enzyme is supplied in:

50 mM Tris-HCl (pH 7.5), 10 mM CaCl2, and 50% (v/v) glycerol.
DNase I activity in the presence of <sup>Mg2+</sup> or Mn<sup>2+</sup>.

DNase I Activity

DNase I activity in the presence of <sup>Mg2+</sup> or Mn<sup>2+</sup>.

DNase I activity in the presence of Mg2+ or Mn2+.


References

  1. J. Sambrook D.W. Russell, Molecular Cloning: A Laboratory Manual The Third edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).
  2. N. Kienzle et al., DNase I treatment is a prerequisite for the amplification of cDNA from episomal-based genes. BioTechniques. 20, 612-616 (1996).
  3. S. Anderson, Shotgun DNA sequencing using cloned DNaseI-generated fragments. Nucleic Acids Res. 9, 3015-3027 (1981).
  4. M. Kunitz, Crystalline desoxyribonuclease; isolation and general properties; spectrophotometric method for the measurement of desoxyribonuclease activity. J. Gen. Physiol. 33, 349-362 (1950).
  5. I. Wiame et al., Irreversible heat inactivation of DNase I without RNA degradation. BioTechniques. 29, 252-256 (2000).

Product Guides and Selectors

pdf   DNase I, RNase-free