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

Figure 3

Another highly sensitive assay that models the use of restriction enzymes is the blue-white color selection assay. In this assay, Stratagenes pBluescript II SK phagemid vector (Figure 3), which contains the lacZ reporter gene, is cut at a single position within the multiple cloning site using a five-fold excess of enzyme. A phenol-chloroform extraction is used to purify the linearized vector. The vector is ligated and transformed into Epicurian Coli XL1-Blue competent cells, which are then plated on a medium containing X-gal and IPTG. Functional b-galactosidase (determined by blue colonies) is produced only for those transformations with plasmids containing uninterrupted reading frames. If the integrity of the DNA ends produced during the digestion reaction is compromised (i.e., a nucleotide is deleted), the lacZ gene is interrupted, and white colonies result. Enzymes tested in this assay (those commonly used in cloning applications) must produce less than 5% white colonies.

Stability Testing

The unit activity of each restriction endonuclease is confirmed at least once every six months. This stability testing guarantees that enzymes are fully active when delivered to your laboratory. Most Stratagene restriction enzymes are guaranteed to be stable for 18 months from the date of testing.

Conclusions

Stratagene recognizes that high-quality reagents are imperative for molecular biology applications and, therefore, commits to providing an extensive line of rigorously qualified restriction enzymes. We stringently establish the functional purity of every enzyme preparation to ensure optimal performance.


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