Quality Control Overview
Unit Determination: One unit of restriction endonuclease activity is defined as the amount of enzyme required to completely digest 1 µg of substrate DNA in a total reaction volume of 50 µl in one hour using the NEBuffer provided. Incubations are performed in capped microcentrifuge tubes at the appropriate incubation temperature as indicated on the Technical Data Card. Concentrated enzymes are diluted to approximately 1,000 units/ml using the recommended storage conditions before determining their activity.
Quality Controls: The results of all quality control assays are reported on the Technical Data Card provided with each enzyme.
Assay for Nonspecific Endonucleases: To assay for nonspecific endonuclease contamination, each restriction endonuclease is incubated with a supercoiled plasmid substrate lacking the recognition sequence of the restriction endonuclease. A single nonspecific nick in the RF I DNA converts it to the RF II form (nicked circle). Aliquots are incubated with 1 µg of RF I (supercoiled form) DNA in a reaction volume of 50 µl using the recommended NEBuffer. Incubations are performed for 4 hours at the recommended temperature. The two forms are easily distinguished on agarose gels and the percent conversion from RF I to RF II is determined.
Overnight Assay for Nuclease Contamination: All NEB restriction endonucleases are incubated overnight in their recommended NEBuffer with 1 µg of substrate DNA in a volume of 50 µl. The characteristic banding pattern produced by the enzyme in one hour is compared to the pattern produced from an excess of enzyme incubated overnight. A sharp, unaltered pattern under these conditions is an indication that the enzyme preparation is free of detectable levels of nonspecific DNases. The maximum number of units that can be incubated overnight is reported.
Assay for Exonuclease Contamination: All restriction endonucleases are incubated with 1 µg of a mixture of single and double-stranded, 3H-labeled E. coli DNA (200,000 cpm/µg) in a 50 µl reaction volume with the supplied NEBuffer. Incubations are at the recommended temperature for 4 hours. Exonuclease contamination is indicated by the percent of the total labeled DNA in the reaction that has been rendered TCA-soluble. The limit of detectability of this assay is approximately 0.05%.
Ligation of DNA Fragments: DNA fragments are produced by an excessive over-digestion of
substrate DNA with each restriction endonuclease. These fragments are then ligated with T4 DNA Ligase at a
5´ termini concentration of 0.1–1.0 µM. The ligated fragments are then recut with the same
restriction endonuclease. Ligation can only occur if the 3´ and 5´ termini are left
intact, and only those molecules with a perfectly restored recognition site can be recleaved. A
normal banding pattern after cleavage indicates that both the 3´ and 5´ termini are intact
and the enzyme preparation is free of detectable exonucleases and phosphatases. An example using Pst I
is shown to the right.
Blue/White Screening Assay: Enzymes used for cloning applications are tested by an additional quality control, the Blue/White Screening Assay, to determine the integrity of the DNA ends produced after digestion with an excess of enzyme. The assay is performed by digesting an appropriate vector at a unique site within the lacZa gene with a 10-fold excess of enzyme, ligating, transforming and plating on XGal/IPTG/Amp plates. Successful cleavage, ligation and expression of b-galactosidase is a function of how intact its gene remains after cloning. An intact gene gives rise to a blue colony. While an interrupted gene (i.e., degraded DNA end) gives rise to a white colony. All restriction enzymes tested must produce fewer than 3% white colonies in order to be Blue/White Certified.