Given the scenario below what restriction enzymes would be b

Given the scenario below, what restriction enzymes would be best suited? In the cloning vector example below, what restriction enzyme(s) should be used to get the gene of interest (thicker black bar) into the plasmid?

Solution

Answer:

Restriction enzymes EcoRI (E) and HindIII (H) cannot be used since the gene of interest contain these sites.

ApaI cannot be used since the plasmid do not contain this site.

The most efficient way to clone the gene of interest into the plasmid is using XhoI (X). This will insert the gene of interest into the plasmid without loosing any fragment due to internal cleavage (XhoI sites are on two sides of the gene of interest).

But using XhoI for cloning can insert the gene of interest into the plasmid in any orientation. So, we need to screen for the right clones through DNA sequencing to confirm the plasmids containing the gene of interest in the correct orientation.