For using restriction enzymes to digest and
ligate genes of interest into plasmid vectors, it is good practice to check if
your restriction enzyme(s) of choice also cuts at regions other than those
intended. For standard commercial vectors, these usually have a multiple
cloning site (MCS) region containing unique restriction sites where the
corresponding enzyme will only cut. However, if you opt to use an enzyme whose
site is not present in the MCS, you will need to check if your enzyme(s) of
choice cuts anywhere else on the vector.
For your insert, if you decide to engineer
restriction sites onto the 5’ and 3’ ends, it is better to (1) choose sites
that do not cut anywhere in the insert and (2) make sure the restriction site
you are incorporating correspond to what is present in the MCS of the vector.
Some good online tools that are freely
available to use for identifying restriction sites in DNA sequences include:
