Subcloning

The idea behind using the same restriction enzymes is to create complementary sticky ends, which will facilitate ligation later on.

After letting the reaction mixture sit for a set amount of time at a specific temperature (dependent upon the size of the strands being ligated; for more information see DNA ligase), the insert should become successfully incorporated into the destination plasmid.

After a good number of bacterial colonies have grown, they can be miniprepped to harvest the plasmid DNA.

In order to ensure growth of only transformed bacteria (which carry the desired plasmids to be harvested), a marker gene is used in the destination vector for selection.

The mammalian DNA does not come with these restriction sites, so they are built in by overlap extension PCR.

The plasmid is transformed into bacteria and the identity of the insert is confirmed by DNA sequencing.