Product Class: Other

Nucleoside Digestion Mix
Gray property icon for NEBU Gray property icon for incubation temperature 37°

Product Introduction

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.

  • Convenient one-step protocol
  • Digests both DNA and RNA to single nucleosides
  • Low-glycerol formulation significantly reduces glycerol-induced ion suppression during MS analysis
  • See what others are saying about the Nucleoside Digestion Mix
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Catalog # Size Concentration
M0649S 50 reactions

Product Information

Description

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.



Figure 1: Global nucleoside analysis of HeLa DNA following incubation with the Nucleoside Digestion Mix
Absorbance tracing versus time, using Nucleoside Digestion Mix
Representative HPLC chromatogram of individual deoxyribonucleosides obtained from incubation of 1 µg of purified genomic HeLa DNA digested with 1 µl of the Nucleoside Digestion Mix for 1 hour at 37°C. Deoxyribonucleosides were separated by reversed-phase HPLC and detected by UV absorbance at 260 nm.


Figure 2: Global nucleoside analysis of HEK 293 RNA following incubation with the Nucleoside Digestion Mix

Absorbance tracing versus time with inset of 3 to 7 minutes
Representative HPLC chromatogram of individual ribonucleosides obtained from incubation of 1 µg of purified genomic HEK 293 RNA digested with 1 µl of the Nucleoside Digestion Mix overnight at 37°C. Ribonucleosides were separated by reversed-phase HPLC and detected by UV absorbance at 260 nm. The insert shows an expansion of the chromatogram highlighting the detection of modified ribonucleosides.


Figure 3: The activity of the Nucleoside Digestion Mix remains stable even after 50 freeze-thaws

Bar graph of activity versus freeze-thaw cycles
After undergoing multiple freeze-thaw cycles, 1 µl of the Nucleoside Digestion Mix was used to digest 1 µg of λ DNA (NEB #N3011) for 1 hour at 37°C. The nucleoside content of the digested samples was analyzed by LC-MS.
This product is related to the following categories:
Hydroxymethylation Detection and Analysis,
Epitranscriptome Analysis,
DNA Methylation Analysis,
Epigenetics,
RNA Modification,
DNA Modifying Enzymes & Cloning Technologies,

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • NEB # Stored at (°C)
    M0649S -20
    Component Name Component # Stored at (°C) Amount Concentration
    Nucleoside Digestion Mix M0649SVIAL -20 1 x 0.05 ml 50 reactions
    Nucleoside Digestion Mix Reaction Buffer B0649SVIAL -20 1 x 0.5 ml 10 X

Properties & Usage

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. 

FAQs & Troubleshooting

FAQs

  1. What applications is the Nucleoside Digestion Mix best used for?
  2. Does the Nucleoside Digestion Mix digest ssDNA?
  3. Does the Nucleoside Digestion Mix digest RNA?
  4. Does the Nucleoside Digestion Mix digest DNA or RNA in a DNA/RNA hybrid?
  5. How much DNA or RNA can the Nucleoside Digestion Mix digest?
  6. What types of epigenetic modifications can be identified using the Nucleoside Digestion Mix?
  7. Are there any nucleic acid modifications that the Nucleoside Digestion Mix cannot digest?
  8. Is the Nucleoside Digestion Mix sensitive to salt?
  9. Will the Nucleoside Digestion Mix digest DNA or RNA stored in TE buffer?
  10. Is it necessary to purify my DNA/RNA substrate prior to digestion with the Nucleoside Digestion Mix?
  11. Is it necessary to cleanup my digested nucleoside mixture prior to LC-MS analysis?
  12. What is the activity of the Nucleoside Digestion Mix in other buffers?
  13. Does the Nucleoside Digestion Mix require ZnCl2?
  14. Can the Nucleoside Digestion Mix be incubated with substrate for >1 hour?
  15. Why does the Nucleoside Digestion Mix freeze when stored at -20°C?
  16. Can the Nucleoside Digestion Mix be stored at 4°C?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

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]

Certificate Of Analysis

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]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

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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