Enzimas de Restrição

Restriction endonucleases

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.