Restriction Endonucleases: Quality Control

Unit Determination

One unit of restriction endonuclease defined as the amount of enzyme required to digest 1 μg of substrate DNA in a total reaction volume of 50 μl in 1 hour using the optimal SE-Buffer provided.

Quality Controls

The results of all quality control assays are reported on the Certificate of analysis provided with each enzyme.

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 FblI see on the figure.

16-hour assay for nuclease contamination

All SibEnzyme restriction endonucleases are incubated for 16 hours in optimal buffer with 1 μg of substrate DNA in a volume of 50 μl. The characteristic DNA fragments pattern produced by the enzyme in 1 hour is compared to the pattern produced from an excess of enzyme incubated for 16 hours. 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 for 16 hours is indicated.

Assay for exonuclease and phosphatase contamination

All restriction endonucleases are incubated for 3 hours with 5'-[32P]-labeled synthetic oligonucleotides (single-stranded and double-stranded) in a volume of 20 μl. After incubation of these labeled oligonucleotides with an enzyme, denatured reaction products are separated on a polyacrylamide gel and then analysed by phospho-imaging. No detectable degradation of single-stranded and double-stranded oligonucleotides indicates that the enzyme preparation is free of exonuclease and phosphatase contamination.