New England Biolabs, M0649S, Nucleoside Digestion Mix

The Nucleoside Digestion Mix is an optimized mixture of enzymes that provides a convenient one-step method to generate single nucleosides from DNA or RNA for quantitative analysis by liquid chromatography-mass spectrometry (LC-MS), eliminating the need for sequential multi-step, time-consuming digestion protocols. Related Categories Hydroxymethylation Detection and Analysis,, Epitranscriptome Analysis,, DNA Methylation Analysis, Specification 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 FAQ Q: What applications is the Nucleoside Digestion Mix best used for? A: The main application of the Nucleoside Digestion Mix is identification and global quantification of DNA and RNA modifications (e.g., methylation) by LC-MS analysis. Q: Does the Nucleoside Digestion Mix digest ssDNA? A: Yes, the Nucleoside Digestion Mix will digest ssDNA into individual nucleosides. Q: Does the Nucleoside Digestion Mix digest RNA? A: Yes, the Nucleoside Digestion Mix will digest RNA molecules (except for mRNA cap structures) into individual nucleosides. The Nucleoside Digestion Mix will also degrade both the DNA and RNA strand of a DNA/RNA hybrid. Q: Does the Nucleoside Digestion Mix digest DNA or RNA in a DNA/RNA hybrid? A: Yes, the Nucleoside Digestion Mix will digest both the DNA and RNA strand of a DNA/RNA hybrid into individual nucleosides. Q: How much DNA or RNA can the Nucleoside Digestion Mix digest? A: The Nucleoside Digestion Mix (1 µl) can effectively digest 1 µg of DNA or RNA to nucleosides in 1 hour at 37°C. Q: What types of epigenetic modifications can be identified using the Nucleoside Digestion Mix? A: The Nucleoside Digestion Mix will digest nucleic acids containing epigenetically and epitranscriptomically modified bases such as 5-methylcytosine, 5-hydroxymehtylcytosine, 5-formylcytosine, 5-carboxymethylcytosine, N6-methyladenine, 5-hydroxymethyluracyl, and many others. The Nucleoside Digestion Mix will also digest certain unnatural or damaged bases. Q: Are there any nucleic acid modifications that the Nucleoside Digestion Mix cannot digest? A: Yes, the Nucleoside Digestion Mix cannot digest an inverted 5´-ppp-5´ bridge (such as in m7G-G mRNA cap structures), thymine dimers or phosphothioesters. The Nucleoside Digestion Mix has reduced activity on oligonucleotides that have highly modified backbones with one or more substituents at the 2´, 4´ or 5´ ribose positions, such as antisense RNAs or other therapeutic oligonucleotides that have been engineered to resist nuclease cleavage. To ensure complete digestion, samples containing complex secondary structures and 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. Alternatively, the ratio of Nucleoside Digestion Mix:nucleic acid may be increased from 1 μl/μg substrate to 5-10 μl/μg substrate. Q: Is the Nucleoside Digestion Mix sensitive to salt? A: The Nucleoside Digestion Mix is relatively insensitive to salt content, however it is recommended that the final salt concentration in the digestion reaction remain ≤ 250 mM. Q: Will the Nucleoside Digestion Mix digest DNA or RNA stored in TE buffer? A: The Nucleoside Digestion Mix will digest DNA or RNA stored in typical TE buffers (≤1 mM EDTA). Unless the final EDTA concentration in the digestion reaction is > 1 mM, it is not necessary to exchange the buffer prior to digestion with the Nucleoside Digestion Mix. Q: Is it necessary to purify my DNA/RNA substrate prior to digestion with the Nucleoside Digestion Mix? A: As carryover of certain reaction components (e.g., EDTA, detergents, etc) may result in incomplete digestion, it is highly recommended that DNA or RNA substrates be purified (column purification or phenol/chloroform extraction followed by ethanol/isopropanol precipitation) and resuspended in water before digestion with the Nucleoside Digestion Mix. Q: Is it necessary to cleanup my digested nucleoside mixture prior to LC-MS analysis? A: It is usually not necessary to clean up the reaction after digestion with the Nucleoside Digestion Mix and prior to LC-MS analysis. Q: What is the activity of the Nucleoside Digestion Mix in other buffers? A: In order to achieve the highest activity possible with the Nucleoside Digestion Mix, we recommend using the supplied Nucleoside Digestion Mix Buffer, however, the Nucleoside Digestion Mix is active over a wide range of pH values (optimally between pH 4.0–7.0) and is also salt tolerant (up to 250 mM). Additionally, since the Nucleoside Digestion Mix requires ZnCl2, reaction buffer should be free of any chelating agents (i.e., EDTA) and supplemented with 1 mM ZnCl2 in order to optimize activity in a buffer other than the supplied Nucleoside Digestion Mix Reaction Buffer. While the Nucleoside Digestion Mix is active in NEBuffer r1.1 supplemented with 1 mM ZnCl2, the Nucleoside Digestion Mix is not active in NEBuffer r2.1, r3.1 or rCutSmart Buffers. NEBuffer r1.1 r2.1 r3.1 rCutSmart® % Activity 100 0 0 0 Q: Does the Nucleoside Digestion Mix require ZnCl2? A: Yes, the Nucleoside Digestion mix does require ZnCl2 and therefore can be inhibited/inactivated by the addition of EDTA. Q: Can the Nucleoside Digestion Mix be incubated with substrate for >1 hour? A: Yes, to ensure complete digestion, the Nucleoside Digestion Mix can be incubated with sample for longer time periods. No signal deterioration has been observed by incubating DNA or RNA samples with the Nucleoside Digestion Mix for longer durations (overnight) at 37°C. Samples containing a large number of modifications (particularly modifications at the 2´-position) may benefit from overnight incubation with the Nucleoside Digestion Mix in order to achieve complete digestion. Q: Why does the Nucleoside Digestion Mix freeze when stored at -20°C? A: In order to reduce the ion suppression effects of glycerol during mass spectrometry analysis, 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. Q: Can the Nucleoside Digestion Mix be stored at 4°C? A: Yes, the mix is stable for at least 2 years when stored at 4°C without significant activity loss. However, we recommend -20°C for long term storage of the Nucleoside Digestion Mix since, as with many aqueous buffers, long term storage at 4°C or higher temperatures may result in bacterial or fungal growth.