Ensuring the quality and performance of NEB’s enzymes is of paramount importance, both to you, our customer, and to us. All of our restriction enzymes undergo stringent quality controls (below), and we are constantly testing their performance through our internal research programs. The result is a consistently superior restriction endonuclease that delivers on its promised performance and activity.
NEB extensively quality controls its popular lines of standard and high-fidelity (HF®) restriction enzymes. Each new lot is tested and must meet the specifications designated for the product. Quality controls for restriction enzymes, both standard and HF, include:
Enzymes are evaluated by SDS-PAGE and silver-stained gels to ensure the highest levels of purity and the absence of contaminating proteins.
Exonuclease Activity:Using radioactively labeled DNA substrate and/or state-of-the-art capillary electrophoresis-based assays with fluorescently labeled substrates, NEB is able to detect very low levels of exonuclease activity.
To ensure that there are no contaminating enzymes that could cause nicking or non-specific nuclease degradation, reagents are incubated with supercoiled plasmid DNA for 4 hours to demonstrate the absence of endonuclease contamination.
Non-specific DNase Activity:
Enzymes are incubated overnight with Lambda DNA to confirm that there is no additional non-specific nuclease activity present.
Cloning QC (Ligation & Re-cutting):
A DNA template is overdigested by the appropriate restriction enzyme, and the percentage of DNA fragments ligated and re-cut are determined by agarose gel electrophoresis.
Cloning QC (Blue-white Screening):
A DNA plasmid is over-digested by the appropriate restriction enzyme, and the linearized plasmid DNA is ligated and transformed into an E. coli strain with greater than 99% correct transformants, as determined by a blue-white screen.
Restriction Enzyme Competitor Study: Nuclease Contamination
EcoRI, NotI, and BamHI from multiple suppliers were tested in reactions containing a fluorescent labeled single stranded, double stranded blunt, 3’overhang or 5’ overhang containing oligonucleotides. The percent degradation is determined by capillary electrophoresis and peak analysis. The resolution is at the single nucleotide level.