Product Class: Kit

5´ DNA Adenylation Kit

Product Introduction

  • This kit contains Mth RNA Ligase (NEB #M2611A)
  • Ideal for generating high yields of 5´ adenylated DNA
  • Easy to use protocol with >95% efficiency
  • One step reaction with no need for gel purification
Catalog # Size Concentration
E2610S 10.0 reactions 50 µM
E2610L 50.0 reactions 50 µM

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Product Information

Description

The 5´ DNA adenylation Kit is a simple and efficient enzymatic method for generating 5´-adenylated DNA. The kit is optimized to produce the adenylated DNA with or without 3´-terminator. The 5´ DNA adenylation kit routinely generates greater than 95% conversion of pDNA to AppDNA(1). This highly efficient process eliminates the need for gel isolation of the product and increases overall yield.
This product is related to the following categories:
RNA Ligation

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E2610S     -20    
      Mth RNA Ligase M2611AVIAL -20 1 x 0.02 ml 50 µM
      5' DNA Adenylation Reaction Buffer B2610SVIAL -20 1 x 1 ml 10 X
      Adenosine 5' Triphosphate N0757AVIAL -20 1 x 0.1 ml 1 mM
  • E2610L     -20    
      Mth RNA Ligase M2611AAVIAL -20 1 x 0.1 ml 50 µM
      5' DNA Adenylation Reaction Buffer B2610SVIAL -20 1 x 1 ml 10 X
      Adenosine 5' Triphosphate N0757AVIAL -20 1 x 0.1 ml 1 mM

Properties & Usage

Features

  • One step reaction gives quantitative adenylation
  • Simpler than existing chemical and enzymatic methods
  • Reduces need for extensive purification of reaction product
  • 65°C reaction temperature reduces secondary structural concerns
  • Easily scalable from pmol to μmol range

Application Features

Enzymatic 5´-adenylation of single-stranded DNA linkers for next generation sequencing.

Product Notes

  1. The adenylation reaction can be scaled up to 6X without a loss of efficiency, to a final concentration of 30 pmol of oligonucleotide and 30 pmol of Mth RNA Ligase per µl. The oligonucleotide can be purified by phenol extraction and alcohol precipitation or column chromatography to remove protein and ATP.
  2. For substrates with unprotected 3´ termini increase concentration of ATP to 0.5 mM to prevent circularization and concatemerization.
  3. The low turnover of the enzyme requires an approximately equimolar concentration of the enzyme and the oligonucleotide substrate.
  4. Adenylated DNA linkers can be used for 3´-end ligation of RNA in cDNA library preparation for Next Generation sequencing protocols [3,4].

References

  1. Zhelkovsky, A.M. and McReynolds, L.A. (2011). Nucl. Acids Res. 39(17): e117.
  2. Torchia, C., Takagi, Y. and Ho, C.K. (2008). Nucleic Acids Res. 36, 6218-6227.
  3. Hafner, M. et al. (2008). Methods. 44, 3-12.
  4. Vigneault, F., Sismour, A.M. and Church, G.M. (2008). Nature Methods. 5, 777-779.

Protocols, Manuals & Usage

Protocols

  1. Protocol for Oligonucleotide Adenylation (NEB #E2610)
  2. Protocol for one-step adenylation and ligation of 5´p DNA and 3’OH RNA using Mth RNA Ligase (NEB #M2611) and Thermostable 5’ App DNA/RNA Ligase (NEB #M0319)

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. Can this 5' DNA adenylation kit be used for adenylating ssRNA oligos?
  2. What is the molecular weight of Mth RNA Ligase? Is it a fusion protein?
  3. What ratio of enzyme to oligonucleotide do you recommend
  4. Can Mth RNA Ligase be used in other NEBuffers?
  5. Can Mth RNA Ligase be used for ligation?
  6. Does PEG stimulate adenylation?
  7. What concentration of ATP should be used in the adenylation reaction?
  8. Will the enzyme work at low temperature (e.g., 37°C)?
  9. How do I determine the extent of adenylation?
  10. Do I need to clean up the adenylation reaction before I use the oligo for ligation?
  11. What is the composition of the 5´ DNA Adenylation Reaction Buffer at 1X concentration?

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 [email protected].

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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