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DNase I, RNase-free

#EN0521 #EN0523 Supplied with: 10X Reaction Buffer with MgCl2 25 mM EDTA	1000 u (1 u/µl) HC, 1000 u (50 u/µl) 1 ml 1 ml
#EN0525 Supplied with: 10X Reaction Buffer with MgCl2 10X Reaction Buffer w/o MnCl2 100 mM MnCl2 25 mM EDTA	1000 u (1 u/µl) 1 ml 1 ml 1 ml 1 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EN0521, #EN0523, #EN0525 MSDS (English) MSDS (English-USA) MSDS (German)

 

Feature
Possibility to select the action mode of enzyme by choice of buffer containing either MgCl₂ or MnCl₂.

Description
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⁺ and Mn²⁺ ions:

• in the presence of Mg²⁺, DNase I cleaves each strand of dsDNA independently, in a statistically random fashion (1), see picture below;

• in the presence of Mn²⁺, the enzyme cleaves both DNA strands at approximately the same site, producing DNA fragments with blunt-ends or with protruding termini of only one or two nucleotides in length (1).

DNase I, RNase-free activity.

Source
Bovine pancreas.

Molecular Weight
31 kDa monomer.

Applications

• Preparation of DNA-free RNA (1).
• Removal of template DNA following in vitro synthesis of RNA with T7, T3, SP6 RNA Polymerases (1), see Protocol for the Removal of Template DNA Following in vitro Synthesis of RNA.
• Preparation of DNA-free RNA prior to RT-PCR (2),  see Protocol for Preparation of DNA-free RNA Prior to RT-PCR.
• DNA labeling by nick-translation in conjunction with DNA Polymerase I (1), see Protocols for DNA Labeling by Nick-translation.
• Studies of DNA-protein interactions by DNase I footprinting (1).
• Generation of a library of randomly overlapping DNA inserts. Reaction buffer containing Mn²⁺ is used (3).

Quality Control
The absence of ribonucleases and proteases confirmed by appropriate quality tests. Functionally tested for digestion of template DNA after in vitro synthesis of RNA using T7 RNA Polymerase.

Concentration
1 u/µl
50 u/µl, HC

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 pBR322 DNA.

Storage Buffer
The enzyme is supplied in: 50 mM Tris-acetate (pH 7.5), 10 mM CaCl₂ and 50% (v/v) glycerol.

10X Reaction Buffer with MgCl₂
100 mM Tris-HCl (pH 7.5 at 25°C), 25 mM MgCl₂, 1 mM CaCl₂.

10X Reaction Buffer without MnCl₂
100 mM Tris-HCl (pH 7.5 at 25°C), 1 mM CaCl₂.
Recommended concentration of MnCl₂ in 1X reaction buffer is 10 mM.

Inhibition and Inactivation

• Inhibitors: 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 50-100 mM.
• Inactivated by heating at 65°C for 10 min in the presence of EGTA or EDTA or by phenol/chloroform extraction.

Related Products

RNA Polymerases: SP6 RNA Polymerase T3 RNA Polymerase T7 RNA Polymerase DNA Polymerase I, E,coli M-MuLV Reverse Transcriptase RevertAid™ M-MuLV Reverse Transcriptase RevertAid™ H Minus M-MuLV Reverse Transcriptase RiboLock™ RNase Inhibitor First Strand cDNA Synthesis Kit  RevertAid™ First Strand cDNA Synthesis Kit  RevertAid™ H Minus First Strand cDNA Synthesis Kit  0.5 M EDTA, pH 8.0 Water, nuclease-free DEPC-treated Water

References

1. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
2. Kienzle, N., et al., DNase I treatment is a prerequisite for the amplification of cDNA from episomal-based genes, BioTechniques, 20, 612-616, 1996.
3. Anderson, S., Shotgun DNA sequencing using cloned DNaseI-generated fragments, Nucleic Acids Res., 9, 3015-3027, 1981.
4. Wiame, I., et al., Irreversible heat inactivation of DNase I without RNA degradation, BioTechniques, 29, 252-256, 2000.

Updated kovo 18, 2008 09:01