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Ligase Quality

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Ensuring the quality and performance of NEB’s enzymes is of paramount importance, both to you, our customer, and to us. Ligases that are optimally pure function as promised, and that is what we strive daily to deliver. Therefore, we have aligned our DNA ligase production methodologies with an expansive series of quality controls (below), and are constantly testing our DNA ligases in house with our internal research programs. The result is a consistently superior DNA ligase product that delivers on its promised performance.

NEB brings a variety of quality controls to bear on its popular line of DNA ligases and ligase master mixes. 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. Quality controls for the DNA ligases and ligase master mixes include:
  • 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.
  • Non-Specific DNase Activity (16 hour): The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
  • Protein Purity (SDS-PAGE): The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
  • RNase Activity (2 Hour Digestion): The product is tested in a reaction containing a RNA substrate. After incubation for 2 hours there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.
  • RNase Activity (16 Hour Digestion): The product is tested in a reaction containing a RNA substrate. After incubation for 16 hours there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.
  • Functional Test (Ligation and Transformation): The product is tested for ligation and transformation efficiency using DNA containing blunt or cohesive termini. Ligation products are transformed and must exceed specifications for efficiency (transformants/µg).

Extreme purity with NEB's T4 DNA Ligase

T4_DNA_Ligase
Equivalent amounts of protein were loaded and silver stained using SilverXpress™. Marker M is NEB’s Broad Range Protein Marker (NEB #P7702).

T4 DNA Ligase Competitor Study - Nuclease Contamination

T4DNALigase_purity_thumb_0415
T4 DNA Ligase from multiple suppliers was tested in reactions containing a fluorescent labeled single stranded, double stranded blunt, 3’overhang or 5’ overhang containing oligonucleotides. The percent degradation by contaminating nucleases is determined by capillary electrophoresis and peak analysis. The resolution is at the single nucleotide level.

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