| Quality Assurance Tests for Restricition Endonucleases |
| Unit Definition |
| One unit of restriction endonuclease is defined as the amount of enzyme required to produce a complete digest of one microgram of appropriate substrate DNA (or fragments) in 60 minutes in a total reaction mixture of 50μl under specified assay temperature and conditions. |
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| Unit Determination Assay |
| Serial discrete enzyme dilutions are prepared using specified storage buffer of the enzyme. A series of unit determination reaction is initiaited by adding 5μl aliquots form the enzyme dilution to 1μg of the appropriate substrate DNA in a total volume of 50μl. The reactions are terminated after 60 minutes incubation at the specified temperature by either heating the sample at 70℃ for 10 minutes or adding 10μl of 6X concentrated stop solution containing 0.1M EDTA. The exact number of units is determined by ascertaining the complete digestion of the substrate DNA by agarose gel electrophoresis. For accurate discrimination between fragments of partial digestion and fragments of complete digestion, different site, concentration and types of gel electrophoreses are performed. |
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| Overdigestion Assay |
| The absence of non-specific endonucleases and exonucleases is confirmed by overdigestion assay. In the overdigestion assay, increasing amounts of enzyme are added to a series of reaction tubes containing substrate DNA. After 16-hour incubation at specified temperature, the maximum number of units giving a clear, sharp and normal banding pattern is determined by agarose gel electrophoresis. The highest number of enzyme units producing an unaltered banding pattern is reported on the data section of a certificate of analysis supplied with each product. |
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| Ligation/Recutting Assay |
| Intactness of the fragment termini generated through digestion of DNA with restriction endonuclease is confirmed by ligation of the fragments with T4 DNA ligase and recutting of the ligated fragments with the same restriction endonuclease. DNA fragments produced by 2 to 100-fold overdigestion with each restriction endonuclease are ligated with T4 DNA lieges at appropriated temperature (16℃ or 22℃) for 4 to 16 hours and then re-cut with the same restriction endonuclease. The extent of ligation and banding pattern after re-cutting are analyzed by gel electrophoresis. Only those molecules with the intact termini can be ligated and generate normal banding pattern of re-cutting. |
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| Other Assay |
| There are so many kind of experiments existed, applicable to the usage of restriction enzymes and modifying enzymes. Our products have been verified and is famous to be suitable, excellent and superior to competitor’s products for most typical experiments like as the construction of cloning vectors, PCR amplification, the construction of transformant strains, the digestion and ligation of DNAs & plasmids, and miscellaneous laboratory works related to molecular biology experiments. In addition, we guarantee you the quality of state-of-art packing & delivery and the lowest price in the market. |
