  Convenient one-step protocol
  Digests both DNA and RNA to single nucleosides
  Low-glycerol formulation significantly reduces glycerol-induced ion suppression during MS analysis

The Nucleoside Digestion Mix is a mixture of enzymes that provides a convenient one-step method to

generate single nucleosides from DNA or RNA. Optimized for quantitative analysis by liquid

chromatography-mass spectrometry (LC-MS), this reagent eliminates the need for sequential multi-step,

time-consuming digestion protocols. The Nucleoside Digestion Mix digests ssDNA, dsDNA, DNA/RNA

hybrids and RNA(except mRNA cap structures) containing epigenetically modified (m5C, hm5C, f5C,

ca5C, m4C, m6A, etc.), unnatural, or damaged bases. Moreover, the low-glycerol formulation (<1%)

significantly reduces glycerol-induced ion suppression during mass spectrometry analysis.

1X Nucleoside Digestion Mix Reaction Buffer.   Incubate at 37°C.

-20°C

1.  Dilution of the Nucleoside Digestion mix may result in a decrease of enzymatic activity and incomplete

      digestion of substrate. Therefore, we recommend using 1 µL of the Nucleoside Digestion Mix for

      digestion of samples containing < 1 µg of DNA or RNA substrate.
2.  Samples containing a large number of modifications (particularly modifications at the ribose 2´-position)

     may benefit from overnight incubation with the Nucleoside Digestion Mix in order to achieve complete

     digestion. No signal deterioration has been observed by incubating DNA or RNA samples with the

      Nucleoside Digestion Mix for up to 24 hours at 37°C. Alternatively, the ratio of Nucleoside Digestion

      Mix:nucleic acid may be increased from 1 μl/μg substrate to 5-10 μl/μg substrate to ensure complete

      digestion.
3.  Although it is not necessary to stop the reaction prior to LC-MS, the mix can be inactivated by the

     addition of EDTA (5 mM, final concentration).
4.  In order to reduce the ion suppression effects of glycerol, the Nucleoside Digestion Mix has been

     formulated to contain very little glycerol (<1%) and therefore the mix will freeze when stored at -20°C.

     The mix is stable for >2 years when stored at -20°C and can withstand 50 freeze-thaw cycles without

     significant activity loss.
5.  As carryover of certain reaction components (e.g., EDTA, detergents, etc) from upstream steps may

     result in incomplete digestion, it is highly recommended that DNA or RNA substrates be purified

     (column purification/phenol chloroform extraction) before digestion with the Nucleoside Digestion

     Mix.