Digestion of DNA with restriction endonucleases



EX: For a 20 ul total volume of reaction (15 ug DNA)


         5 ul DNA: Maximum DNA 1/3 of total volume

         2 ul Buffer 10X: Maximum 1/10 of total volume

         2 ul enzyme: NEB maximum 1/10 of total volume

         11ul H20: Total volume – (DNA, Buffer, and Enzyme Volume)



Example: BGLII, 0.25 Units, so 1ml has 10,000U, then 1 ul has 10 units.


1ul of BGLII will digest 40ug of DNA


How should I set up a restriction digest?


A2: Most researchers follow the general rule that 10 units of restriction endonuclease is sufficient to overcome variability in DNA source, quantity and purity. Generally, 1 μl of enzyme is added to 1 μg of purified DNA in a final volume of 50 μl of the appropriate 1X NEBuffer followed by incubation for 1 hour at the recommended temperature. If an excess of enzyme is used, the length of incubation can often be decreased to save time. Alternatively, you can productively digest with fewer units of enzyme for up to 16 hours with many restriction enzymes.

To keep glycerol concentration at less than 5% in a reaction, the restriction enzyme, which is supplied in 50% glycerol, should not exceed 10% of the total reaction volume.

An extremely important, yet often overlooked, element of a successful restriction digest is mixing. The reaction must be thoroughly mixed to achieve complete digestion. We recommend gently pipetting the reaction mixture up and down or “flicking” the reaction tube. Follow with a quick (“touch”) spin-down in a microcentrifuge. Do not vortex the reaction.


Q8: Is extended digestion (incubation times > 1 hour) recommended?


A8: The unit definition of our restriction enzymes is based on a 1 hour incubation. Incubation time may be shortened if additional units of restriction enzyme are added to the reaction. Conversely, longer incubation times are often used to allow a reaction to proceed to completion with fewer units of enzyme. This is contingent on how long a particular enzyme can survive (maintain activity) in a reaction. Some enzymes survive for long periods (> 16 hours) while others survive only an hour or less in a reaction. For each restriction enzyme, we report the minimum number of units (1.0, 0.5, 0.25 or 0.13) required to digest 1 µg of substrate DNA in 16 hours. Enzymes that require less than 1 unit can be used at lower concentrations for extended incubation times. Note that DNA substrates are digested at varying rates, the actual number of units required for a complete digestion will change from substrate to substrate. Check individual restriction enzyme information before extending reaction times, as those that exhibit star activity should be used under recommended conditions to inhibit noncanonical cleavage.


 FROM : http://www.neb.com/nebecomm/products/faqCategory1.asp


This entry was posted in Protocols. Bookmark the permalink.