Restriction Enzymes Resource 

2.5 Digestion of Supercoiled Plasmid DNA

Restriction enzyme units are usually defined using linear DNA substrates containing multiple recognition sites as these tend to give more reproducible results. Lambda and Adenovirus are the two substrates used most frequently because of their commercial availability and high quality. One unit of enzyme is the amount necessary to completely digest 1µg of such DNA in one hour under the appropriate buffer and temperature conditions.

Molecular biology applications frequently involve cutting a supercoiled plasmid at a single site within the multiple cloning sequence. Often, more than 1 unit of enzyme is required to digest 1µg of plasmid. There are several reasons why this is the case. For example, there are 0.0317 picomoles of DNA in 1µg of lambda. Hind III cleaves this substrate 7 times or 0.2219 picomoles of recognition sites in 1µg. For a 3,000 base pair plasmid with a single recognition site, there are 0.5 picomoles of DNA in 1µg and also 0.5 picomoles of recognition sites, over twice as many as for the same mass of lambda DNA. The ability of a restriction enzyme to find a single site by linear diffusion in the supercoiled plasmid is also presumed to be different than for any of the sites on a linear substrate. Although it is not common, some enzymes exhibit differences in their ability to cut supercoiled DNA depending on the buffer conditions used. For example, Sac II exhibits a pronounced difference in its ability to cut supercoiled plasmids depending on buffer conditions, but this sensitivity is not seen nearly as dramatically with linear substrates. Promega's Reaction Buffer C, supplied with Sac II works well for both linear and supercoiled DNA substrates.

Table 2.5 lists the minimum number of units necessary to completely cut 1µg of a supercoiled pGEM^® Vector^(a) containing a single recognition site. Commonly used Promega restriction enzymes, including all those that are blue/white cloning qualified, are listed.

^(a)U.S. Pat. No. 4,766,072 has been issued to Promega Corporation for transcription vectors having two different bacteriophage RNA polymerase promoter sequences separated by a series of unique restriction sites into which foreign DNA can be inserted.

pGEM is a trademark of Promega Corporation and is registered with the U.S. Patent and Trademark Office.

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