Restriction Endonucleases: Quality Control
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.
The results of all quality control assays are reported on the Certificate of analysis provided with each enzyme.
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.