Restriction+Digest+Protocol

Basic outline for a restriction digest: may want about 20 uL volume with 500 ng plasmid, BSA, Buffer, water, 1 uL enzyme; if performing a sequential digest move from lower to higher salt;

Note that if a vector has been digested, and a ligation will be performed, then the alkaline phosphatase reaction to prevent self ligation can be performed right away in the same tube. Then all of the enzymes can be removed, and the correctly sized fragment can be purified in a gel purification reaction. see Promega CIAP Protocol. Also note that in this situation it may be a good idea to let the cut vector run on the gel for a long time to make sure that all of the cut vector is well separated from the uncut vector.

Note that when doing a sandwich gel extraction after a restriction digest it may be a good idea to run two totally separate identical gels so that it is as easy as possible to align everything.

Resources
 * [[file:fermentas buffer conversion chart.pdf]]