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Restriction Enzymes
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| SE Buffers and Activity |
Unit Determination:
One unit of restriction endonuclease activity is defined as the amount of enzyme required to completely digest 1 µg of substrate DNA in a total reaction volume of 50 µl in one hour using the buffer provided. Incubations are performed at the appropriate incubation temperature as indicated on the Technical Data Sheet.
Quality Controls:
The results of all quality control assays are reported on the Technical Data Sheet provided with each enzyme.
Ligation of DNA Fragments:
DNA fragments are produced by the 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. A normal banding pattern after cleavage indicates that both 3´ and 5´ termini are intact and the enzyme preparation is free of detectable exonucleases and phosphatases.
Overnight Assay for Nonspecific Nucleases:
All restriction endonucleases are incubated overnight in the recommended buffer with 1 µg of substrate DNA in a volume of 50 µl. The characteristic banding pattern produced by the enzyme in one hour is compared to the pattern produced by the excess of enzyme incubated overnight.
