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:
- Physical Purity:
Enzymes are evaluated by SDS-PAGE gel to ensure the highest levels of purity and the absence of contaminating proteins.
- Endonuclease 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.
- 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.
- Functional Test (15 minute Digest):
A 50 μl reaction containing 1 μg of DNA and 1 μl restriction enzyme incubated for 15 minutes results in complete digestion as determined by agarose gel electrophoresis.
- DNA Contamination (E. coli Genomic):
Enzymes are screened for the presence of E. coli genomic DNA using SYBR® Green qPCR with primers specific for the E. coli 16S rRNA locus.