• My NEB
  • Print
  • PDF
  • 5´ DNA Adenylation Kit

    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.

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Adenosine 5' Triphosphate1 mM
    5' DNA Adenylation Reaction Buffer10X
    Mth RNA Ligase

    Advantages and Features

    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.

    Applications

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

    Properties and Usage

    Storage Temperature

    -20°C

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Phosphatase Activity (PNPP):
      The product is tested in a reaction containing a p-nitrophenyl phosphate (PNPP), a chromogenic substrate for most phosphatases. After incubation there is no detectable phosphatase activity as determined by spectrophotometric analysis of released p-nitrophenol at 405 nm.
    • RNase Activity (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    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.

    Supporting Documents

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Protocol for Oligonucleotide Adenylation (E2610)

    Selection Tools