DNase I (RNase-free)

For research use only.
Please centrifuge briefly before opening (volume ≤2 ml).

Description:
DNase I (RNase-free) is a highly purified recombinant bovine endodeoxyribonuclease expressed in yeast. The enzyme nonspecifically cleaves single- and double-stranded DNA, producing mono- and oligodeoxyribonucleotides with 5'-phosphate and 3'-hydroxyl termini.

DNase I activity is dependent on Ca²⁺ and is activated by Mg²⁺ or Mn²⁺ ions. In the presence of Mg²⁺, the two strands of double-stranded DNA are cleaved independently at statistically random sites. In the presence of Mn²⁺, both strands are cleaved at approximately the same site, producing blunt-ended fragments or fragments with overhangs of one or two nucleotides.

Unit Definition: One unit is defined as the amount of enzyme required to completely degrade 1 μg of pBR322 DNA in 10 minutes at 37 °C under the specified assay conditions. One unit of DNase I corresponds to 0.3 Kunitz units.

Shipping: shipped on gel packs

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles

Shelf Life: 12 months

Form: liquid

Concentration: 5 units/μl

Applications:
DNase I (RNase-free) is used to remove contaminating genomic or plasmid DNA from RNA preparations, degrade DNA templates following in vitro transcription, and reduce viscosity caused by genomic DNA in cell lysates. It is suitable for applications in which efficient DNA degradation without damage to RNA is required.

Content:
DNase I (RNase-free)
5 units/μl DNase I supplied in 10 mM Tris-HCl pH 7.6 (25°C), 2 mM CaCl₂ and 50 % [v/v] glycerol

DNase I Reaction Buffer
10 x conc.

Reaction Conditions
For preparation of DNA-free RNA, use 1 unit of DNase I per μg of RNA in 1 x DNase I Reaction Buffer and incubate at 37°C for 15 min.
High levels of monovalent ions such as Na⁺ and K⁺ (i.e. 100 mM) may decrease DNase I activity.

Inactivation:
Add EDTA to a final concentration of 2.5 - 5.0 mM and heat at 65°C for 10 minutes to stop the reaction.
The sample can be directly used for downstream applications such as reverse transcription.

Activity:
DNase I activity is also measured in Kunitz units. One Kunitz unit is defined as the amount of enzyme required to produce an increase in absorbance of 260 nm of 0.001/min/ml at 25°C of highly polymerized DNA.
1 unit of DNase I is equivalent to 0.3 Kunitz units.

Selected References:
Kunitz, M. (1950) Crystalline desoxyribonuclease. I. Isolation and general properties. J. Gen. Physiol. 33: 349–362.
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. New York: Cold Spring Harbor Laboratory Press 10.6
Pan et al. (1999) Ca2+- dependent activity of human DNase I and its hyperactive variants. Protein Sci. 8:1780.