M0649S, Nucleoside Digestion Mix - 50 reactions

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.

Supplied in: 20 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 2 mM CaCl2, 2 mM ZnCl2, 50 mM NaCl and 0.6% glycerol.

• Reaction Conditions

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

1X Nucleoside Digestion Mix Reaction Buffer
50 mM sodium acetate
1 mM ZnCl₂
(pH 5.4 @ 25°C)

Storage Buffer

20 mM Tris-HCl
1 mM MgCl₂
2 mM CaCl₂
2 mM ZnCl₂
50 mM NaCl
0.6% Glycerol
pH 7.5 @ 25°C

• Product Notes
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.