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Restriction Endonucleases Undergo Rigorous QC Testing to Ensure Optimal,,,Performance

esentative sample from each packaging of a restriction endonuclease is confirmed prior to shipment to demonstrate that no loss of enzymatic activity occurs upon packaging.

Overdigestion of Substrates

When enzymes are tested under standard digestion conditions (1 U of enzyme/g substrate DNA), trace levels of contaminating DNase activities might not be detected. To more stringently assess the functional purity of each enzyme preparation, Stratagene incubates a 4- to 100-fold excess of enzyme with substrate DNA. After a 16-hour incubation at the appropriate temperature, the banding pattern from these overdigested samples is compared to the signature-banding pattern produced by the enzyme in 1 hour. If the restriction endonuclease preparation is free from contaminating endonuclease and exonuclease activities, the overdigested reactions will exhibit the identical banding pattern characteristic of the 1-hour reactions. The maximum number of units that clearly exhibit this pattern in the overdigestion reactions is reported on the Certificate of Analysis provided with each enzyme. This overdigestion assay is also employed on a representative sample following aliquoting and packaging to confirm that contaminating DNase activity is not introduced during packaging.

Detecting Exonuclease Contaminants

The integrity of a restriction enzyme cut site can be destroyed by the presence of exonucleases that can digest single-or double-stranded ends. Stratagene specifically assays for exonuclease activity by incubating an excess of restriction endonuclease for 4 hours with a mixture of single- and double-stranded, [3H]-thymidine-labeled genomic E. coli DNA. Contaminating exonuclease is indicated if over 0.6% of the 3H-labeled DNA added to the reactions is detected as trichloroacetic acid-soluble counts.

Ligation Assay

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