DNase I-XT
NEBU 37 No

  • Salt-tolerant DNA-specific endonuclease
  • Degrades double-stranded and single-stranded DNA
  • Generates short oligos with 5′-phosphate and 3′-OH
  • RNase-free

DNase I-XT is ideal for:

  • Degradation of DNA templates from in vitro transcription reactions
  • Removal of contaminating genomic DNA from RNA samples
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Catalog # Concentration Size
M0570S 2,000 units/ml 1,000 units
M0570L 2,000 units/ml 5,000 units
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  • Product Information
    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.


     
    This product is related to the following categories:
    Exonucleases and Non-specific Endonucleases Products,
    DNA Modifying Enzymes & Cloning Technologies Products
    This product can be used in the following applications:
    RNA Analysis,
    RNA Purification and Isolation
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