DNase I

Product Description

• DNase I is a DNA-specific endonuclease that cleaves double-stranded and single-stranded DNA to oligonucleotide products with 5'-phosphate and 3’-OH.  The enzyme is expressed in E. coli as a recombinant 6His-tagged fusion protein, and the 6His-tag together with the fusion are removed during purification.

• DNase I is free of RNase activity.

Applications

• Removal of DNA templates in in vitro RNA transcription reactions.

• Preparation of RNA samples free of genomic DNA contamination during RNA purification or cleanup.

• Controlled DNA digestion in such as DNA fragmentations.

Unit Definition

One unit of DNase I is defined as the amount of enzyme that completely degrades 1 µg of pBR322 plasmid DNA in 10 minutes at 37 °C.

Concentration

2 U / µl

Recommended Reaction Condition

Set up reactions in 1X DNase I Reaction Buffer with 2 U (1.0 µl of MDAD019) and incubate at 37 °C for 15 min. Add 0.5 M EDTA to a final concentration of 5 mM (~1.5x higher than the concentration of total divalent ions in the reaction) and incubate at 85 °C for 10 minutes to inactivate DNase I before downstream applications. When necessary, dilute samples to avoid high salt concentration which is known to inhibit DNase I activity. There is no significant inhibition of activity at < 50 mM salt and  ~50% activity at 100 mM salt.

Key Performance Data

Rapid Degradation of DNA by DNase I

Time course of DNA degradation by DNase I. DNase I digestion reaction was set up with 1 µg of pBR322 plasmid DNA in standard reaction buffer with 0.25, 0.5, 1.0 or 2.0 U of DNase I and incubated at 37 ℃ for 5, 10 or 15 min. The reactions were stopped by adding 10 mM EDTA and then analyzed by 1 % agarose gel electrophoresis. As shown, even 5 min digestion was able to degrade 1 µg of pBR322 plasmid DNA. In our protocol, we recommend 15 min digestion time to assure completely removal of DNA.

Suitable to Remove DNA in RNA Applications

DNase I is free of RNase activity on single-stranded (A) or double-stranded RNA (B). RNA template were treated with 1, 2 or 4 U of  DNase I in standard digestion buffer in 20 μl at 37 ℃ 15 min. After inactivation of the DNase I, 5 μl of the reaction was mixed with equal volume of 2 x RNA loading buffer and analyzed in a 1% TBE agarose gel.  The RNA remain intact in all reactions.