Biotechnology Index Glossary
Cutting DNA With Restriction Endonucleases
A large number of restriction enzymes are commercially available, and one of the best sources of information about their characteristics and conditions of use is to be found in the reference sections of supplier's catalogs or on their web sites.
For a first-time user, at least three items deserve emphasis regarding restriction digestions:
- Restriction enzymes are expensive (some much more than others)
- Restriction enzymes are, in general, heat labile: Enzymes should be kept at -20C except for brief periods of time on ice or in a small freezer box. It is distressingly easy to leave a little box of enzymes on the lab bench overnight and lose several hundred dollars.
- Contaminating one enzyme with small quantities of another can cause massive confusion and loss of time to yourself and your coworkers: Never pipet an enzyme with anything other than a new pipet tip!
Setting up a simple restriction digestion is easy - there are three mandatory ingredients that need to end up in the same tube:
- DNA: Reliable cleavage by restriction enzymes requires DNA that is free from contaminants such as phenol or ethanol. Excessive salt will also interfere with digestion by many enzymes, although some are more tolerant of that problem.
- An appropriate buffer: Different enzymes cut optimally in different buffer systems, due to differing preferences for ionic strength and major cation. When you purchase an enzyme, the company almost invariably sends along the matching buffer as a 10X concentrate.
- The restriction enzyme! Don't laugh - at some point in your career, you'll set up a group of digests, forget to add the enzyme, and wonder why nothing cut.
The amount of DNA to be used depends on the task at hand, but for the sake of example, consider a typical diagnostic digestion to confirm the identity of a plasmid:
In this case, 1 microgram (ug) of DNA should suffice and 20 ul would be a convenient volume for the reaction.
After deciding which enzyme to use, look in the supplier's catalog to determine which buffer and incubation temperature is recommended. Into a microcentrifuge centrifuge pipet 2 ul of 10X buffer, the DNA and enough water to bring the total volume to 20 ul. Add the enzyme last - most are provided at a concentration of 10 to 20 units per ul, and 1-2 units would suffice for this type of digestion.
It is important to mix the reaction, which is easily done by "flicking" the tube and then shaking down its contents. Incubate the reaction at the recommended temperature for 30 to 60 minutes, then analyze by gel electrophoresis.
The senario described above is meant as an overview of a typical reaction. There are many variations on the theme and factors that influence enzyme activity. Another common practice is to include in the reaction a small quantity of a protein like bovine serum albumin, which often enhances the reaction, particularly when the DNA is not exceptionally pure.
Next Topic for Biology and Activity of Restriction Endonucleases: Restriction Mapping Advanced and Supplemental Topics
Last updated on October 20, 1999
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