Fragment of interest is inserted into a plasmid cut by the restriction
enzyme EcoRI. A series of restriction digests are run on the
new plasmid, such that each enzyme is used in a separate digest, then both
are used together along with a fragment size marker or DNA "ladder".
The disappearance of bands from each single digest and the appearance of
new bands in the double digest suggests that one of the enzymes (in this
example, BamHI) has cut within the
In #2 above, the EcoRI digest has separated the inserted DNA from the original vector. The insert is 6580 bp in size. In the double digest, a fragment 1740 bp in size has been cleaved from the insert, while a fragment 1120 bp has been removed from the vector (the entire BamHI fragment is 1740 bp + 1120 bp = 2860 bp; see the gel above). By using multiple digests, it is possible to construct a restriction map, which can then be used to find the smallest possible fragment of insert DNA containing the gene of interest. That fragment can then be removed from the gel, ligated into a new plasmid, and transformed into E. coli. This is called subcloning. Large genes having more that 40,000 bp. must be maintained as several clones in E. coli.