Restriction enzymes are enzymes that cut both single- and double-stranded DNA. Each restriction enzyme has a specific nucleotide sequence, called a restriction site, that it recognizes and cuts. Restriction enzymes are used for DNA sequencing, mutational analysis, and cloning and amplification of DNA. Scientists use restriction enzymes to insert genes of interest into expression vectors, DNA molecules that can replicate separately from chromosomal DNA. The vector containing the gene of interest can then be introduced into a bacterial strain for expression and characterization of proteins.
Identify restriction enzyme sites on your vector by looking at a restriction map. The restriction map will tell you which enzymes will cut your vector, and where.
Choose a restriction enzyme that also has a site present on your gene insert, by looking at the sequence of the insert. Ensure the restriction site is at a position on your insert that is outside the gene of interest, so you do not lose any part of the gene.
Ensure that there are no duplicates of the restriction site anywhere in your gene insert or vector. This will cause multiple cuts in your DNA and give you misleading data.
Try to choose restriction enzymes that cut with sticky ends, rather than blunt ends. Sticky ends occur when the enzyme cuts double stranded DNA in a staggered manner, leaving a single stranded overhang that facilitates attachment with an insert cut in the opposite manner. Blunt ends occur when the double-stranded DNA is cut in a smooth manner, and these are more difficult to attach.
Choose a different restriction enzyme for both ends of your insert to ensure it is inserted into the vector in the proper orientation and to ensure the vector does not re-attach to itself.
Try to choose two restriction enzymes which function well in the same buffer system and temperature. If this is not possible, run each digestion separately.