Stratagene confirms the functional purity of each restriction enzyme preparation using an assay that simulates a typical cloning protocol. In this assay, substrate DNA is completely digested using a three-fold excess of enzyme, and the enzyme is removed using StrataClean resin. The digestion products are ligated using Stratagenes T4 DNA ligase and then recut with the same restriction enzyme. Samples of the cut, ligated, and recut DNA are subsequently analyzed by agarose gel electrophoresis (Figure 1). If the ends of the digested DNA have been compromised by contaminating exonucleases or phosphatases, the recognition site will no longer be intact, and the cut product will not ligate efficiently or, when recut, yield a banding pattern identical to the initial digest. Stratagene reports the percent of ligated and recut substrates on the Certificate of Analysis.
To analyze for contaminating endonuclease or topoisomerase activities, Stratagene incubates an excess of restriction endonuclease with supercoiled plasmid DNA that lacks the restriction enzyme recognition sequence. Following a 4-hour incubation at the appropriate temperature, the conversion of supercoiled DNA (RF I) to the open circular form (RF II) is monitored by agarose gel electrophoresis (Figure 2). This conversion is indicative of a single nick in the RF I DNA. If greater levels of contamination are present, linear DNA (RF III) might also be observed. Stratagene guarantees that its restriction endonuclease preparations will generate <5% conversion to RF II DNA and no detectable conversion to RF III DNA.