How much restriction enzyme?

Sometimes, you need to know how much restriction enzyme is required to cut a specific amount of a certain plasmid within a given time. Here is the calculation tool you have been looking for! All data that was used to write the code for this algorithm was obtained from New England Biolabs (NEB). The survival of the enzyme in the reaction was extrapolated from the experiments reported by NEB. Just plug in the numbers into the form and hit the submit button!

Remember that many other factors do increase the amount of enzyme needed, such as the buffer, degree of supercoiling, etc. This calculator is more meant to give you a ballpark idea of the amounts you'll need. It cannot account for all the variables that influence an IRL digest. Do not forget that these days, restriction enzymes can get quite old in the fridge, and they tend to lose activity over time. That also means that a newly bought restriction enzyme is usually overprovisioned, i.e., its real activity is a bit higher than what is indicated on the tube. As a vendor, you typically want to have a safety margin; nothing is worse than a customer complaining about a dead enzyme. The number calculated here is something like the minimal amount required under optimal conditions. If you have made the mistake of buying FastDigest enzymes from Thermo Fisher (TF), this calculator cannot help you since TF does not report their activity. Your best bet is to stick to a reputable vendor with complete transparency, such as NEB. The tool assumes that the DNA operates in its recommended buffer at its recommended temperature. Please note that I collected the data from NEB about 25 years ago when I was a PhD student. I have never updated them since. I have now just ported the code into Drupal without doing any modifications. If I find some time, I might update the data and perhaps incorporate buffer usage into the tool (which would help with double digests). However, TF fails again on the transparency front, since many of their buffer compositions remain a trade secret. Hence, it would require an effort to incorporate TF buffers into that tool.