An engineered variant of DNase I, DNase I-XT is a salt-tolerant DNA endonuclease that nonspecifically cleaves DNA to release di-, tri- and oligonucleotide products with 5′-phosphorylated and 3′-hydroxylated ends (1,2). DNase I-XT acts on single- and double-stranded DNA, chromatin and the DNA strand of RNA:DNA hybrids. While DNase I (RNase-free) (NEB #M0303) is inhibited by salt concentrations >50 mM, DNase I-XT (NEB #M0570) exhibits optimal activity between 50-100 mM salt and retains 65% and ~40% activity in 200 and 300 mM salt, respectively. This increased salt tolerance makes DNase I-XT the preferred enzyme for DNA template removal from an in vitro transcription (IVT) reaction. Importantly, DNase I-XT is RNase-free, allowing for the complete removal of DNA from RNA preparations while maintaining RNA integrity.
Figure 1: DNase I-XT efficiently degrades DNA at salt concentrations > 100 mM
An equimolar comparison of the DNase activity of DNase I (NEB #M0303) and DNase I-XT (NEB #M0570) illustrates the increased salt-tolerance of DNase I-XT as compared to DNase I. DNase activity was measured by an increase in fluorescence from a quenched 35 nt hairpin dsDNA substrate in 1X DNase I Reaction Buffer with increasing salt concentration
(as indicated). While DNase I activity steadily decreases with increasing salt concentrations, DNase I-XT remains active in solutions containing up to 300 mM salt.
Figure 2: DNase I-XT removes more DNA from IVT reactions and RNA preparations
In vitro transcription reactions (20 μl) were treated with 1) no DNase I; 2) 2 U TURBO® DNase or 3) 2 U DNase I-XT for 15 minutes at 37°C. Each sample was then purified using the Monarch® RNA Cleanup Kit (500 µg, NEB #T2050) and eluted in nuclease-free water (50 μl). The level of residual DNA contamination was quantified by real-time PCR using the Luna® Universal Probe qPCR Master Mix (NEB #M3004). Average Cq (quantification cycle) values for each sample were compared to a standard curve (gray) to determine the percent of residual, PCR-amplifiable DNA. Both TURBO DNase and DNase I-XT require no dilution of the IVT reaction prior to DNase digestion, however, more DNA template is removed from an IVT reaction and undetectable by qPCR when treated with DNase I-XT.
Figure 3: DNase I-XT efficiently removes residual genomic DNA from crude RNA preparations
Crude RNA samples were subjected to either: 1) No DNase treatment; 2) in-solution treatment with DNase I-XT (2 U, NEB #M0570) in DNase I-XT Reaction Buffer; 3) in-solution treatment with DNase I (2 U, NEB #M0303) in DNase I Reaction Buffer or 4) in-solution treatment with TURBO DNase (2 U, ThermoFisher) in TURBO DNase Reaction Buffer for 15 minutes at 37°C. Residual genomic DNA (gDNA) was quantified (+/– RT) by RT-qPCR using the Luna Universal One-Step RT-qPCR Kit (NEB #E3005) and human or mouse actin exonic primers. DNase effectiveness was calculated by the difference between the average Cq value -RT (amplified DNA) and the average Cq value +RT (amplified DNA and RNA).
DNase I-XT removes more contaminating gDNA from crude RNA preps than either DNAse I or TURBO DNase.
Product Source
A His-tagged engineered variant of DNase I expressed in Pichia pastoris.
This product is related to the following categories:
One unit is defined as the amount of enzyme required to release 210 pmol of FAM from a 35mer FAM-BHQ1 labeled hairpin oligonucleotide in 1 minute at 30°C in a 50 µl reaction volume.
Reaction Conditions
1X DNase I-XT Reaction Buffer
Incubate at 37°C
1X DNase I-XT Reaction Buffer
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
0.5 mM CaCl2
0.005% Tween® 20
(pH 7.6 @ 25°C)
Storage Buffer
10 mM Tris-HCl
2 mM CaCl2
50% Glycerol
pH 7.6 @ 25°C
Heat Inactivation
No
Advantages and Features
Features
Use in reactions containing higher amounts of salt, such as IVT and RNA preps
Add directly to your IVT reaction, with no dilution required
Efficiently removes DNA from IVT reactions and RNA preps
RNase-free enzyme tolerates a wide range of salt conditions (up to 300 mM)
DNase I-XT is supplied with an optimized reaction buffer for DNase I-XT. For optimal activity, we recommend using DNase I-XT (NEB #M0570) with DNase I-XT Reaction Buffer (NEB #B0570) and not with DNase I Reaction Buffer (NEB #B0303). Likewise, due to the salt in DNase I-XT Reaction Buffer being inhibitory to DNase I, we do not recommend use of DNase I-XT Reaction Buffer (NEB #B0570) with DNase I (NEB #M0303).
We do not recommend using NEB's DNase I-XT as a substitute for Monarch DNase I in the RNA isolation workflow when using the Monarch Total RNA Miniprep Kit (NEB #T2010). Monarch DNase I is optimized for use in this workflow, and is available for purchase as a component of the Monarch Total RNA Enzyme Pack, (NEB #T2019).
References
Kunitz, M. (1950). J. Gen. Physiol.. 33, 349-362.
Vanecko, S. and laskowski, M. (1961). J. Biol. Chem.. 236, 3312-3316.
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Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
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