Restriction Digestion

So, you have your DNA plasmid, and you want to see if your insert DNA is truly in the plasmid or not.  What needs to be done is a restriction digestion, where the plasmid is cut with a restriction enzyme (at least twice; if you cut a circular piece of DNA just once, you still have the same size DNA, just linearized).  The point of this exercise is to show that the insertion was successfully made, so to test this there should be a restriction site within the insert DNA, and a site in the vector itself.

A key fact to remember when doing the restriction digestion is that you need roughly 200ng (nanograms) of DNA fragment to visualize it on an electrophoresis gel. Because if this, careful calculations must be made so that you get enough fragment to visualize.

In the above example, the vector itself is 2 kilobases; the fragment is 200 basepairs. Because of this:

1 microgram of plasmid = 100 nanograms of insert (for this particular case)

Thus, we need to use at least two micrograms of plasmid to produce enough fragment to visualize.

The second thing to take into account is the restriction enzyme being used.  Restriction enzymes are usually distributed fairly concentrated (10-15 units/microliter, where one unit is enough to cut one microgram of DNA per hour).  If the restriction enzyme provided is diluted down, again, calculations must be done in order to assure adequate enzyme and/or time for the plasmids to be cut.  Note also that environmental factors such as salt concentration, temperature, and glycerol concentration may play roles in restriction enzyme activity.

Typical restriction digestion “recipe” is as follows:

X microliters of DNA
Y microliters of water
2 microliters of 10x buffer
1 microliter of enzyme
----
20 microliter reaction

Where X is the amount of DNA you need to obtain over 200 nanograms of fragment, and Y is the amount of water needed to take the reaction to the 20 microliter total.