Specific Activity: ≥ 2000 Kunitz units/mg
Form: Lyophilized Powder
Storage: 4°C
DNase Ⅰ (RNase-free) is an endonuclease that nonspecifically cleaves DNA to release di-, tri- and oligonucleotide products with 5´-phosphorylated and 3´-hydroxylated ends. DNase I acts on single- and double-stranded DNA, chromatin and RNA:DNA hybrids.
Feature
- Recombinant enzyme
- Purified from non-animal host with a lower level of intrinsic RNases.
Applications
- Degradation of DNA template in transcription reactions
- Removal of contaminating genomic DNA from RNA samples
- DNase I footprinting
- Nick Translation
Quality Control
The absence of ribonucleases is confirmed by appropriate quality test. Functionally tested for digestion of template DNA after in vitro transcription
Source: A E. Coli strain that carries an MBP fusion clone of Bovine Pancreatic DNase I.
Molecular Weight: 29 kDa monomer
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.
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 (use at least 1 mol of EGTA/EDTA per 1 mol of Mn2+/Mg2+).
Note: DNase I is sensitive to physical denaturation. Mix gently by inverting the tube. Do not Vortex.
Preparation Instructions
DNase I is soluble in 0.15 M NaCl at 5 mg/ml. Solutions of DNase I at 5 mg/ml in 0.15 M NaCl may lose 10% of its activity when stored for a week in aliquots at -20°C. The same solutions stored in aliquots at 2-8°C can lose 20% activity.
Alternatively, solutions at a minimum concentration of 1 mg protein/ml, with 5 mM calcium chloride and stored at -20°C, may retain 90% activity for at least a year. Solutions containing 0.1 mg protein/ml are considerably less stable, and may require gelatin as stabilizer.
For applications unaffected by glycerol, two other storage buffers are options, as these formulations do not freeze at -20°C.
- 20 mM sodium acetate (pH 6.5), containing 5 mM CaCl2 and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 50% (v/v) glycerol, with DNase I at 5 mg/ml.
- 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, 50% (v/v) glycerol, with DNase I at 2 mg/ml.
